qPCR troubleshooting: interpreting amplification curves and diagnosing problems

I have a question. 
 
In the problem of "baseline drift", what's the rational of "remove DTT from your reverse transcription step"? 
 
Actually, when I remove DTT, the degradation of probe was stopped.  
It worked. But how??

Hi, just 1 quick question, how to remove DTT? I use ambion PCR reagents.

Thanks

DTT is a strong reducing agent capable of reducing the azo linkages that are present in BHQ® (Black Hole Quencher®) dyes. Reduction of the azo bond in the BHQ dye results in a loss of quencher activity and thus releases fluorescence from the probe, which appears as background observed in the baseline drift. To review chemical structures, feel free to visit our BHQ modification webpages located here: https://www.biosearchtech.com/products/custom-oligonucleotides/oligo-modifications/quencher-labels-for-custom-oligo-synthesis.

I have a question regarding the jagged signal. I check my qPCR product on the gel and got the correct size, no primer dimers. What would be the other possible reasons?

A jagged signal in your amplification plot is typically the result of poor signaling of the probe. The jagged appearance is the result of the program magnifying the baseline noise to compensate for the low signal. There are several potential causes for low signal. One possibility is that the concentration of the probe is too low. Another may be that the melting temperature of the probe is marginal. When the Tm is low, probe binding is weak and signal release is diminished. You may also need to consider the length of the probe. If the distance between the fluorophore and the quencher dye is too great, then inefficient quenching and elevated background fluorescence is expected. It is important to recognize that the fluorophore selection can have a profound impact on the performance of the probe. Some fluorophore and quencher dye pairs are known to form intramolecular dimers, forming a pseudo-beacon structure that is very efficiently quenched. In a very robust assay switching dyes is usually seamless, with only a slight change in cycles to threshold (CT or Cq) value. Such variation is typically on the order of 1 to 2 cycles and relates to the differences in dye intensity as well as the variation in instrument optics across the different channels. Fundamentally, all real-time thermal cyclers are engineered to detect fluorescein (FAM) first and foremost, so dyes with longer wavelength emission may be detected less sensitively.

Hi, I also have a question. 
In my dissociation curve, my unknown samples are showing a melting peak at a lower temperature than my positive controls. These are not primer dimer (because the peaks do not coincide with the peaks of the no-template-control). I don't think it's unspecific product because my bands in the gel are the correct length. This is an environmental sample- so the gene I am amplifying could be slightly different than the positive control gene. But would this make such a big difference in the melting temperature of the product? My positive controls melt at 92 degrees and the unknown samples are melting at 75.5 degrees with a much broader peak. (I am looking at a graph of Rn'(T)). 
Thanks!

Sybr Green dissociation curves include all possible double-stranded products. If your target product has a Tm of 92 degrees then you definitely are not amplifying only your target. It may be that you have some amplification of your target but that the amount is very limited and so it is hidden under tail the larger 75.5 non-specific peak. If any amplification of your target has occurred then you will get a band in your gel.  
 
Are you aware of shorter splice variants? 
What does your amplification plot look like? Does it more closely resemble a linear amplification instead of exponential amplification? 
 
To limit your experiment to include only the target sequence, set the software to collect data at only 92 degrees. Be sure to allow for a long extension time around 72 degrees as I suspect your target is quite long and you may have incomplete extension of the product. Collection at 92 degrees will capture ½ of the amount of product as this represents the temperature at which 50% of the product is dissociated.  
 
Also keep in mind we don't support Sybr Green products, and we do suggest trying our BHQ probes instead for future experiments.

Difference in the slope value of the standard curve could be due to inhibitors in the sample also right?? 
 
Poor PCR efficiency

Yes, inhibitors in the PCR reaction may cause the efficiency to change. These inhibitors may be carryover contaminants from the RNA or DNA isolation steps. To determine if an inhibitor is present in template, a careful review of the Ct values at the higher dilutions compared to those at lower dilutions, will show that the more dilute the sample the more closely the Ct values fit the desired serial dilution. Diluting away the contaminant is not always an option and in these situations you may try ethanol precipitation or other DNA purification method.

We are doing single cell PCR and have observed very different fluorescent plateau heights for different primers or even the same primer on different samples (the plot is X-axis cycle, Y-axis deltaRN). Any idea what may be causing this, if it's normal, or how to fix it?

Variability in the fluorescent plateau heights with high copy number transcripts is often a result of insufficient amounts of reaction components, i.e. dNTPs, leading to a lower plateau and lower final yield. In that situation you would repeat the assay using new reagents or a different master mix. As you are doing single cell PCR you are most likely interested in genes with very low copy number and are already using a master mix specially formulated for high efficiency amplification. One factor which may affect the fluorescent plateau height is the amount of background fluorescence. High baseline signal is an indication that the probe is not efficiently quenched. This may be determined by reviewing the raw data (without baseline subtraction) to see if there is a significant increase in the baseline fluorescence in those assays with lower fluorescence plateaus. If this is the case, then the issue is likely related to probe degradation. As this issue appears to be randomly occurring, it may be due to a contaminant carried over from sample preparation or a degradative agent that was introduced during assay set up, to some but not all preparations.

I am making standard curves for my templates and I notice that after a certain dilution the Ct values either go lower or are plateauing. What is the reason why the Ct values plateau after the concentration of template is too low? What exactly is happening at this point?

When running a Sybr Green assay and dilutions of the template become too low, the formation of primer-dimers (a non-specific product) may occur. The low template samples may present with Cq values that are lower than anticipated based on the Cq values of samples at higher concentration, just as you have described. The best way to determine what is going on is to look at the melting curves of the no template control (negative control or water blank) and low template samples in comparison to a higher template sample to see if a non-specific product is being amplified either alone or in conjunction with the desired template. Typically, primer-dimers have a significantly lower melting temperature and present with a low, broad melting curve peak.  
 
If detecting cDNA, please note that contamination with genomic DNA will lower the Cq value of low template samples and will signal as product in the No Reverse Transcription (no RT) control. It is essential that you run a positive control, negative control, no RT control and water blank when evaluating a new or modified assay design. 

i put the qpcr reaction when i analyse the results it shown base line error in all the wells

Hi Ashish, we will need some more information to help you troubleshoot the problem. Could you email techsupport@biosearchtech.com a screen capture of your amp plot and let us know which instrument you’re using?

Hai i have a question 
When i did QPCR all my ct values are same i.e., 26. I have water treated, DMSO treated and small molecule treated sample sets for the analysis. It suppose to show difference in the wild type and mutant for a particular gene. previously when i did i got a good difference but last time it not happened don't know the reason. Can i get a good suggestion. Thankyou in advance.

Hi Dharamdeep, techsupport will follow up on your question through the email you provided.

Dear sir, 
I use to set gene copy number determination by taking plasmid having single copy of GOI for standard preparation. For standard prepartion, should I aliquoted plasmid DNA or use directly from stock ? If I am aliquoting the plasmids, should I again quantify the plasmid before setting of the reaction or not ? 
If I am aliquoting and storing the plasmid DNA, what would be the frequency of adsorbtion of plasmid DNA on the inner wall of storage vial ?

Hi Hitarth, if you will be using these standards repeatedly, it is probably best to prepare aliquots of your plasmid. This will prevent repeated freeze thaw cycles which could degrade your standards over time. I would recommend preparing aliquot volumes with just enough material for one day’s worth of experimentation. 
 
If the dilutions are prepared and quantitated prior to aliquot than repeated measuring of each aliquot is probably not necessary. However, it does not hurt to be extra diligent in the preparation and quantitation of standards.  
 
As to your last question about absorption of material, you should store samples and standard material in low-retention molecular grade plastics. Binding of nucleic acid to these tubes should be minimal and not be appreciable to your qPCR assay or generation of standard curve. If this remains a concern, consider using a carrier (like yeast tRNA 100 ng/uL) to prevent drop-out (presumably adsorption) of the more dilute standards.  

Hi! I was wondering if you have any advice as to what level of consistency you can expect between assays when using aliquots of a plasmid standard. The highest concentration of my standard curve (1E+08 copies per well) has Ct values that range from about 9-12 cycles between different assays, even when the R^2 values of my standard curves are good and my technical replicates are consistent. Do you happen to have any guidelines in terms of how variable your Ct values for plasmid standards should be between assays, or what level of variability you should be concerned about? Any help is greatly appreciated!

Hello Christina,

To have a better understanding of the real issue we would need all the details, including the data along with the standards, which devices and fluorophore are used, the protocol, the primers/probe sequences, etc.

However, generally speaking, you should not have wide ranges of Ct values for your plasmid standards. You might want to double check the reconstitution/dilution of your oligos and standards. Pipetting errors may sound like a no brainer but could be easily overlooked. You might also consider creating a standard curve that spans a lower concentration range and see if that helps. Finally, if plasmids are vortexed too enthusiastically they could be damaged. This seems less likely but is still a possibility.

Regards,
Hsiao Yuan

Hi Hsiao Yuan,

Thanks for getting back to me! I really appreciate the suggestions for how we can trouble shoot this issue. I also noticed in this thread that James Flynn advised Petya (below) to manually set the threshold to avoid introducing variance; is this something that we should be doing as well? We have been using the automatic setting.

Thanks,
Christina

Hi Christina,

You are welcome. Yes, it is not the best practice for qPCR to let the software select the threshold value. The software will often pick an inappropriate value which will result in spurious results. It is more appropriate to use the same threshold across repeated runs of the same assay, and to do this it is necessary to assign the threshold manually. If you let the instrument’s software automatically choose the threshold every time, then it will assign a different threshold every time, because the software does not know that you are running the same assay repeatedly, and it will likely introduced variance from run to run.

Regards,
Hsiao Yuan

Hello, 
I have a question regarding the threshold setting. In a typical experiment, I am running the same cDNA with primers for different target genes, lets say gene A, gene B and housekeeping gene. The software usually sets an automatic threshold for each target gene, which usually differs a lot. Is it advisable to set the same threshold for all target genes or should I keep the automatic setting? I am not comparing the genes with each other, but always normalising gene expression to the expression of the housekeeping gene. 
Thanks a lot! 
Petya

Hello Petra,  
 
I believe your best option is to normalize all your data using the same positive control sample and assay on every plate run. Set the threshold for each assay manually and use these same settings for every run. If you use the auto-threshold option, it will likely introduce variance from run to run as you have already seen. To manually select the initial threshold value for each gene, pinpoint the beginning of the linear phase of the amplification curve. Check to see if your data still has inconsistent Ct/Cp values for your housekeeping gene from plate to plate. If the assay is not performing reliably then something else in your reaction setup is causing the variable results.  

Thanks a lot, James! I will try this out.

I did qPCR by using Sybr green in 20 ul volume. First time I did not get any product. Second time, accidentally I did not change the volume to 20 ul while running qPCR (7500)machine and it remained on 50 ul which is by default. I got the product. Later when I repeated the first qPCR by adjusting volume each time I did not get any product. 
What could be the possible reason? 

Hello Abdul,  
 
Just to be clear, we do not provide Sybr based dyes at Biosearch although we do support oligo design utilizing such dyes. Biosearch Technologies also provides a number of fluorogenic probe assay formats for gene expression and SNP detection. From your post it seems you are using ABI’s 7500, we suggest you consult their devices manual and software help section for the specific adjustments to the reaction settings. From the description of you results, it sounds like either your reaction chemistry is not consistent or the template you are trying to detect is in such low abundance that it is at limit of detection. We would suggest obtaining a robust positive control to run in your reaction, and then use this standard multiple concentrations to test your primer set. If you continue to obtain variable results you might consider redesign of your test with our RealTimeDesign software.  

When I ran replicates of very low input DNA (ie, 10 copies/mL), I occasionally get linear amplification instead of exponential curves (which happened most of the time). What could be possible root causes for the lower and linear curve?

Hi, 
I have an issue with huge variation between Ct values of technical replicates. The desired segment is amplified well but the difference between replicates can be up to 3 cycles. I'm using a TaqMan probe with TET dye and BHQ1 quencher. Primers, pipetting, mastermix are all working No similar issue when using different probe or SYBR assay). Reagent mixing was also sufficient for other probes. 
Could this issue be resolved by lowering primer/probe concentration? I'm using 1 uM and 0,2 uM respectively. 
So far altering the temperature haven't resolved the issue. 
Any ideas? 
Thanks! 

Hello Hans,  
 
Thanks for your post. I do not think the probe and primer concentration is the cause of your issue. Although, you might want to consider lowering their concentration of you primers and probe as they are included in the reaction at a fairly high concentration. Variation in the Ct from well to well usually indicates insufficient mixing of the reagent, but as you say this is not the case in your reaction. Are you also making sure to spin your PCR plate or tubes before running the reaction? Not having all of the fluid in the bottom of the well can also cause the Ct vary. If this is also not the cause of the issue, then it could be the reaction itself. Are your technical replicates amplifying at a very late point in the reaction? The variation could be due to a very small amount of template in the reaction which is causing stochastic variation from well to well. The design of the probe may also provide a variable signal if it is designed to bind at too low a melting temperature. You can see if your probe’s sequence meets our design recommendations using our oligo evaluator in the RealTimeDesign software . If this still does not solve your issues you can always contact us at techsupport@biosearchtech.com and we will work with you to resolve this issue. 

Hi, 
 
I am performing an allele-specific real-time PCR Taqman assay on samples with a known point mutation verified at another lab. Specifically, the assay is designed with 8 separate reactions that include 7 mutant specific forward primers, 1 reference primer, and all with the same reverse primer. A total of 50 cycles are run. My amplification plots confirm the point mutation established by the other lab testing by pyrosequencing; however, I am getting multiple late peaks on my amplification plots that correspond to the allele specific forward primers that are not mutated. Any thoughts?

Hello Michael,  
 
Thank you for your question. Mutation detection is reliant on a number of factors including the prevalence of the mutation in the sample and the efficiency of the assay design. For low frequency mutations in a high background of “normal” sequences offer require multiple rounds of optimization by empirical testing. Unfortunately, I would need some more details of the experiment to provide any more meaningful advice. We would be happy to work with you on this assay to see if we can improve the specificity and sensitivity of the test. Please contact us at techsupport@biosearchtech.com.  

In the qpcr experiments, I have a standard graph consisting of 10 fold dilutions of fetal RNA which has been made into cDNA then amplified. 
 
Some of my fetal RNA samples are more concentrated than my standard and some are more dilute than the standard. Consequently these lie outside the standard graph. 
 
Although we have a standard graph, we are looking at the comparative change in gene expression at different days as the cells age. 
 
My question is that in our case, is it absolutely essential to get all the points on the standard graph in order to compare the gene expression. I mean should we be diluting or concentrating samples just to get the samples on the standard graph since those points that are outside the standard graph are essentially being predicted by the qpcr machine or the program. 
 
Is it recommended that the diluted samples Cq be then multiplied by the dilution factor and similar is the case for concentrated samples? 
 
Please clarify. 

Hi Anil, 
 
We recommend reading over the MIQE Guidelines in reference to your inquiry about the standard curve: 
 
http://www.gene-quantification.com/shipley-miqe-chapter-8-2011.pdf 
 
Starting on page 8 that covers qPCR Validation, it covers setting up a standard curve to determine the linear range of detection for your target assays: 
 
“Depending on the Cq value recorded for this first run, dilute the PCR product 100- to 1000-fold in 100- to 1000-fold in 100 ng/uL E. coli or yeast tRNA(nuclease-free, molecular biology grade) in nuclease-free water. A 7-log range in 10-fold decrements from the initial dilution is usually sufficient to cover a high range down to 1–10 copies of template. From this experiment, you will find the dilution at which the assay no longer functions in a linear fashion with the higher template concentrations. The last dilution where the value was still linear (falls on the standard curve) is the LOD or lowest limit of detection. You can make 2-fold dilutions around this value to get a more accurate value if required. The critical consideration is that no Cq value can be reported if it falls below the LOD of the assay. “ 
 
So you would need to first determine the upper and lower limitations of your target assay using the standard curve. Then you will need to make sure your fetal RNA samples fall within this range so that you are getting an accurate and linear Cq values. So yes, you can dilute your samples to fall within this range.

Hi, 
I have a number of samples which have the jagged lines indicative of no amplification. However my no template control does show a very small amount of amplification (which I would expect). If my NTCs are showing a small amount of amplification how come some of my samples show no amplification at all?

Hello Sarah,  
Thanks you for your question. This could be caused by a number of different things. But ultimately it sounds as if there is little or no signal in some of you samples which the software reads as a jagged signal. However, this is not the case in some of your NTC’s which are reporting a low background signal possibly with some non-specific amplification in the later cycles. The solution could be a simple as a component missing from the reaction or insufficient mixing of the reagents. This could also be indicative of a number of more complex issues causing the signal detection issue. Would it be possible to provide us with a plot of the raw fluorescence signal and any additional information about the reaction conditions such the probe format and component concentrations? Also please provide the sequence of your primer and probe so we can rule out the oligo design as an issue. You can email this information to TechSupport@biosearchtech.com and we would be glad to troubleshoot this issue with your assay via email. Have a great day!  

Hi, 
I would like to duplicate a published qPCR assay for KRAS mutations; however, when entered into UCSC in-silico PCR tool, the published primer sets fail to deliver a product. The in-silico tool produces a product only if the reverse primer is reverse complemented, and even then it is only 45 bp long. The paper says it should be a 104 to 110 bp PCR product (depending on the primer set). Also, the molecular beacon has the same sequence as the reverse primer. Am I missing something here or is there an error in the published table of primer sequences? The WT primer sequences are: 
Forward: GGTAGTTGGAGCTGGTGGC 
Reverse: AGAGTGCCTTGACGATACA 
 
Any help is greatly appreciated.

Hello Michael,  
 
Please address your assay design questions to Tech Support. We would be happy to work through your specific assay issues. Please be sure to also include a link or PDF of the citation which you mentioned in you post. Thank you.

Hi,  
 
I have a confusion and your guidance is appreciated. I run a plate but since I did not get result for some of the samples, I repeated the in the second plate which I was running some other samples for the same gene. So when I want to do a gene study, I cleared the extra samples on the second plate keeping only repeated samples in it. surprisingly the expression numbers are different compared to when I dont clear extra samples in the second plate and just do the gen study for the repeated samples. can you please let me know why this difference happpens? because I assumed that delta delta ct is only depending on the sample and IRC sample but it seems that other samples in the plate also affect the numbers. Thank you.

Hello,  
 
Thanks for your question. It sounds like this might be an issue with the data normalization. Please email  
techsupport@biosearchtech.com and we can try to work through this issue over email.

I have a SYBRgreen assay that was optimized for detecting a specific fungal species. However when I run environmental samples, my samples have a peak at a higher melt temperature than expected. The expected product has a melt peak at 81C, and the samples have a melt peak at 86C. Is there any way to address this problem? Thanks

Hello Mareli, 
 
We don’t know all the details about your assay, however we could suggest a few things that could be going on here. We would need to know if you observing a single peak at a higher melt temperature, or multiple peaks? If you are observing multiple peaks in your melt curve, there could be some contaminant in the reaction or some other off target amplification due to another template in the sample matching those primers. If so, you might consider the addition of a dual-labeled probe as a detection method which is potentially more specific detection method.  
 
The lack of specificity in the signal associated with SYBRgreen can potentially give rise to nonspecific signal for any amplified material. This is in comparison to a dual-labeled probe. The signal from the fluorophore in the unbound state is quenched; signal is only observed when the reporter dye and quencher are physically separated by binding to the target amplicon. Adding a probe specific to the gene of interest might allow you to eliminate the signal from nonspecific products. 
 
A second possibility is that you are only observing the single peak a few degrees above your predicted Tm. If this is the case you don’t have much need to worry, the predicted and empirical Tm can often differ. Tm is influenced by a number of factors and it is very difficult for software programs to take all of these into consideration, as such the calculated Tm is an estimate rather than a defined number. If you have a positive control within the experiment, also giving you a slightly higher than expected Tm, you can confirm that the amplified product is actually the gene of interest. As a secondary precaution measure, you can run the PCR products on a gel to confirm the correct product size as a single band or proceed to sequence the PCR product to confirm the identity of the amplicon.  
 
I hope I have provided enough information to address your current concern. If you would like guidance on a new assay design, please contact tech support with any other questions you may have, and our team would be happy to further assist you. Have a nice day.  

Hai.. I have a doubt.
I have been using SYBR premix ex taq from takara . But after 1 experiment or so, i am finding contamination in the NTC and the signal which is in the NTC can also be seen in other wells too where the template is used. I am totally confused now. Could you please help me with this bizarre situation. I am very much inexperienced in qRT-PCR though i have experiences in performing PCR.
Thank you

Hello Neena,

Thank you for your question. Unfortunately, without more information about your experiment, it is difficult to accurately pinpoint the source of the signal in your NTC. As SYBR Green releases signal upon intercalation into any double-stranded DNA, it is often difficult to distinguish specific amplification of your product from any spurious products generated (such as primer dimers). When using SYBR in your qPCR assay, it is necessary to run a post-amplification melt-curve to determine the Tm of the contaminant. You might also consider running the PCR products on an agarose gel to determine their size. If you are observing a small product in your NTC or products of multiple sizes in your samples, you may need to optimize your primer set to the assay. Increasing the annealing/elongation temperature so that it is higher than the Tm’s of any primer secondary structures could minimize the detection of primer dimers.

Alternatively, you might consider switching to a probe-based detection method, in which signal is only released upon hybridization to the target amplicon. These probes are designed to hybridize to the target sequence between the primers, and this third layer of specificity will eliminate any spurious signal generated. If you would like to pursue this option and require assistance with your probe design, please email techsupport@biosearchtech.com with the details of your experiment, including the target amplicon and your current primer set, and our team would be happy to further assist you.

Please also note that the contaminant could have been introduced into your NTC during the reaction set up. Thus, I would also recommend cleaning your work area and pipettes with a 10% bleach solution in nuclease-free water and replacing your reagents before proceeding. If you have any other questions or would like further assistance, please contact our technical support team. Good luck with your experiments.

Best,
Dru Cowan

Hi,
I'm running SybrGreen qPCR, and have been getting similar Ct values for negative RT controls on certain genes. beta-Actin Ct values for negative RT controls yield nothing though. All my qPCR primers have been primer-blast and are exon-exon boundary spanning (in at least one of the primers). I have also cleaned up my work area, UV my pipette, cleaned them with RNaEasy, ethanol, and changed nuclease-free water. Any suggestions to what the problem may be? Thanks?

Hello Michell,

Thank you for your question. It sounds as though you've already taken a number of measures to ensure that you’re getting amplification of the specific product, so it could be that the signal you are observing is resultant of primer dimer formation or off-target amplification. As SYBR Green releases signal upon intercalation into any double-stranded DNA, it is often difficult to distinguish specific amplification of your product from any spurious products generated. Therefore, it is necessary to run a post-amplification melt-curve to determine the Tm of the contaminant. You might also consider running the PCR products on an agarose gel to determine their size. If you are observing a small product in your negative RT control or products of multiple sizes in your samples, you may need to optimize your primer set to the assay. Increasing the annealing/elongation temperature so that it is higher than the Tm’s of any primer secondary structures could minimize the detection of primer dimers.

With more information about your experiment, our technical support team could better assist you. If you would like further assistance, please email tech support with more details, including the target amplicon, your current primer set, and some graphs of the raw data that you are encountering issues with. Alternatively, you might consider switching to a probe-based detection method, in which signal is only released upon hybridization to the target amplicon. These probes are designed to hybridize to the target sequence between the primers, and this third layer of specificity will eliminate any spurious signal generated. If you would like, our team could also assist you with probe design, provided the target amplicon and primer set. Have a great day.

Hi, I have a question regarding the fluorescence intensity in a qPCR assay using three different polymerases that show differences in in the fluorescence plateau but otherwise having identical Ct values and linearity. Do you have any suggestions? Thanks for your help.

Hello Andreas,

Thank you for your question. There are a few factors that could be contributing to differences in the fluorescence plateaus. Actually, these differences are not necessarily indicative of any problems within your assay. The initial starting amount of template is reflected in the response curve by the Ct value for each sample. Regardless of the initial template concentration in your assay, the curve should saturate and plateau whenever critical components of the reaction become limiting. Thus when comparing different polymerase enzymes, any differences in the saturation point reflect the threshold at which these enzymes became limiting. Such differences in performance are to be expected when using different enzymes, and do not generally present an issue. You might check out the following review paper on qPCR for a more in-depth description:

Kubista, M. et al. The real-time polymerase chain reaction. Mol. Aspects Med. 27, 95–125 (2006). DOI: 10.1016/j.mam.2005.12.007

If you are still concerned about the quality of your assay, email Technical Support (techsupport@biosearchtech.com) with more details about your experiment and some pictures of your amplification curves. Our team can then better address any questions or concerns you may still have. Have a nice day.

Dear sir
I have negative results for sample in qPCR while the same samples give positive curve in the same run assay but just by changing well location can you help me about that. thanks

Hello Yasser,

In a case where the same sample is providing varying results, the assay really just needs to be performed again until the results are repeatable. However, this issue could fall into one of two categories:

1) The positive sample is a false result. If contamination occurred during PCR setup, there is a chance that you are observing a truly negative sample and a false positive sample. In this case, the contamination likely stemmed from a pipetting error. Please make sure to change out pipette tips after each use during the reaction setup in order to prevent cross contamination between samples. Additionally, you may want to clean your reaction setup area and pipettes with a 10% bleach solution to remove any potential contaminants.
2) The negative sample is a false result. In the case that a sample is truly positive but a negative result is obtained, something is missing from the reaction. Most likely, no template DNA was added to the sample well. However, if the master mix is not mixed properly enough, a component of the reaction (primer, probe, etc) could be missing from that sample. Always ensure to thoroughly mix all solutions before pipetting for the most consistent and accurate results.

As long as you follow these standard practices, I expect that this issue would be resolved upon repeating the assay. Please email techsupport@biosearchtech.com if you have any further questions or concerns. Have a nice day.

Dear, Sir
I worked with HCV Viral RNA and worked with onestep RT-QPCR, I want to improve the real time curves mainly the sensitivity of reaction because there are some samples gave me a false negative I' think the rxn volume is the reason because I worked with 15ul Mix and 10 ul Viral RNA.
Regarding to false negative which I faced when I diluted the sample 1:1 I got a positive results.
Recommended me if I increased the rxn volume up to 50 ul we will find a better results and tell me how can I increase the run volume up to 50 ul by increasing the mix by double and sample by double also or recommended me..
Best regards
A. Mohamed

Hello,

Thank you for your question. From the description provided, it sounds as if your assay is potentially resulting in false negatives due to a low copy number of template. The first step in verifying this assay should really be to establish the limit of detection for the assay where you can reliably detect the viral RNA. When the copy number is very low, the amplification of template can become stochastic resulting in false negatives or simply variable amplification. By increasing the reaction volume, you might slightly improve the odds of avoiding this situation by also scaling up the amount of template in the reaction. However, really you need to determine the minimal copy number which can be reliably detected and then use this as a means of qualifying or rejecting data points. You could accomplish this with a serial dilution of known template to form a standard curve (Copy number vs. Ct) and then establish the cutoff for reliable detection. Once this is in place you could possibly explore the option concentrating the target RNA copy number in your sample above this cut-off. If you have further questions or concerns, please contact . Have a nice day.

Hello,

I have a question concerning my amplification curve for a set of primers. When I analyze the melt curve everything looks fine (same with gel analysis). Yet, when I look at the amplification curve I notice that my curves are not only amplifying at higher cycle numbers, but that they are also plateauing at lower RFU values and that the curve begins to dip down after 32 cycles. I'm guessing I might have too much DNA loaded, but I wanted to see if there might be something else wrong.

Thank you for any feedback you might be able to provide,

Tami

Hello Tami,

Thank you for your question. Without knowing how much DNA is being loaded into your reaction, it is hard to be certain if that is the cause of the issue, but indeed it could be. Too much template in the reaction, as well as an excess of primers, can hinder the function of Taq polymerase by limiting the amount of cations available to the enzyme.

You might also want to analyze your primer pair design; if the Tm difference between the primers is too high (>5°C), you might obtain unequal extension and a poor efficiency. There could also be something in the reaction, such as carry over from sample prep or some other contaminate, affecting the reaction efficiency. If other sets of primers are also giving you trouble, the problem might lie in the reaction conditions themselves, so you might have to experiment with the annealing temperature used. If you continue to encounter problems, email techsupport@biosearchtech.com the raw data you are obtaining as well as a description of your reaction conditions and sequences, and we would be happy to further assist you.

I have done some PCR using markers such as VEGF, AT1 and AT2 in substantia nigra from rats.

The samples amplifies very well! I don't have any problem with my samples. However, blanks using beta actin and specific markers don't amplify considering the melting curve for each marker but  there is a non specific amplification. What is the reason for this? Is it possible dimers linking each other? If so, how can I solve this trouble? The primers are new, pipettes are clean before using to begin any procedures for PCR

Hello Clynton,

It sounds as if you are using a DNA binding dye such as Sybr Green for signal detection if you are using a melt curve for assay validation. We support the use of dual-labeled probe based qPCR for signal detection. The use of a target specific probe provides an added layer of specificity to the detection of the target amplification. The DNA binding dye will report signal from any spurious amplification such as primer dimers as you suggested. This can ultimately impact the quality of your data. We would certainly be happy to help troubleshoot your assay at .

Best,

James

Hello Clynton,

looks like the email got cutoff. The address is techsupport@biosearchtech.com.

It sounds as if you are using a DNA binding dye such as Sybr Green for signal detection if you are using a melt curve for assay validation. We support the use of dual-labeled probe based qPCR for signal detection. The use of a target specific probe provides an added layer of specificity to the detection of the target amplification. The DNA binding dye will report signal from any spurious amplification such as primer dimers as you suggested. This can ultimately impact the quality of your data. We would certainly be happy to help troubleshoot your assay at .

Best,

James

Hi again,

looks like the email did show up. The address is techsupport@biosearchtech.com

Hello,
We are using a SYBR assay and see until 1-2 cycles difference between replicates. However, we are using low-retention tips and automatic multidispenser pipets.
qPCR mix are put in the wells then water then samples in triplicates all using muldispenser to ensure accurate volumes. Mixing and spinning of reagents and samples are performed upfront.
We do not want to mix upfront the qPCR mix and samples as we have several samples and are working in plates (multichannel pipetors).
Would you know where could the variation come from with our replicates?

Hello Bea,

This variability in your replicates could come at any number of points in your process of preparing the reaction. There is also certainly some technical variance inherent to the qPCR reaction and data collection itself. Understanding the contribution of variation from the assay design, the detection format, reaction preparation, etc. is critical to drawing any useful conclusions from the data. I might suggest reading a review article by Kitchen et al.(2010) titled "Statistical aspects of quantitative real-time PCR experiment design". I am not certain of your particular experiment but it may be possible that the lack of a sufficient mixing to all the reaction components might contribute to technical variation.

Hello,
I have run the qPCR with SYBR green kit. The exponential phase for negative qPCR sample and the RT negative samples have been started from cycle 30 and more (ct value 35-40) But there is just a negative line for RNA template (no amplification). As the amplification cycles for these negative qPCR and RT negative samples have not been started in the early cycles which could indicate DNA contamination what is the reason for these positive curves in the late cycles.
Thanks,
Elham

Hello Elham,

This sounds like you might have some non-specific amplification in your reaction. This might be from the formation of spurious products generated from primer dimers or possibly some other contaminating nucleic acid which is triggering a late amplification event. Because this is a Sybr based assay the detection via the dsDNA binding dye can not discriminate between the true target amplicon and non-specific product and will report signal from both. LGC Biosearch Technologies specializes in dual-labeled hydrolysis probe based assays which adds a layer of security against signal generation for these off target sequences. We would be happy to assist you further if you contact our technical support team at techsupport@biosearchtech.com.

Best,
James

Hi James,
Thanks for the suggestion. I agree with you about this weakness of SYBR. If I couldn't escape from these non-specific products, I would contact to your technical team.
The confusing event is that, I have also a doggy melting curve. The signals in the lower melting temperature is higher than the amount of signals that I have for the PCR product. As I assumed that these earlier signals of melting temperature are related to the Primer Dimers, I checked the PCR products on the 2% gel electrophoresis. Surprisingly, I don't have neither Primer Dimers not expected PCR product on the gel. It seems that the reaction totally didn't work.
Do you have any idea?

Bests,
Elham

Hello Elham,

It is very difficult to pin point the cause of this phenomenon without actually seeing the data you are observing. Please write in to the technical support team with your data, assay design, and reaction conditions and we should be able to provide some suggestions to resolve this issue. We look forward to hearing from you soon. Have a great day.

Best,
James

Hi James,
Do you think the low fluorescence intensity in the y axis for the house keeping gene might cause a problem?
I am doing a relative qPCR for a gene which has a fluorescence of 20 but the fluorescence for my housekeeping gene which is GAPDH is just around 5. the house keeping amplification still starts at a reasonable cycle I think (Ct 25). There is also a visible band for GAPDH when I ran mu qPCR products on the agarose gel. Do you think i can go with this? what acceptable fluorescence should have a Housekeeping gene? can I go with 5-8?

Thanks alot

Hello Elahe,

The fluorescence units on qPCR devices are arbitrary, and are also specific to the instrumentation and device software itself. If you would like we could have a look at your data and provide suggestions if something looks out of the ordinary. Please send some images of the un-normalized qPCR curves and a description of the reaction conditions to . I look forward to hearing from you.

-Jim

Hi,
Thank you very much for your quick response. I replied to your email as I had to attach some photos. I was wondering if you have received my email. your prompt reply is highly appreciated as I have this experiment left to publish my data.

Thank you very much

Hello Elahe,

My apologies, I should have clarified my response that you should send the data to . If you reply to the Hubspot email it will not reach our inbox. I look forward to hearing from you.

Best,

James

Dear admin,
I had a weird looking curves in my serial dilution samples. The curves from the three lowest concentrations are fine and the sample with the highest conc. is also fine but the curves from the sample with the forth and the fifth highest concentrations have weird peaks. These peaks start at a very early cycles (below the base line and below the reference dye (in this case rox) and as soon as they go over the base line they drop very fast again. unseemlier to the rest of the peaks. The samples are suppose to be treated in the same manner and a multi channel pipett was used for the pipetting. Do you have any idea about how can one interpret the problem?


Cheers

Hello Ammar,

Sorry to hear that you are getting some abnormal results with you dilution series. Please email us at and we would be glad to have a look at your data. I hope that we can find a simple explanation of this issue. Please be sure to provide some images of the raw fluorescence data as an attachment to the email. I look forward to hearing from you. Have a great day.

-James

Hi,
I have problem with my QPCR experiment. I got the same Ct value for the expressed and non-expressed gene. On the agarose elfo there is no band as there is no gene expression. Why I still got the same Ct value as my housekeeping gene. My housekeeping gene highly expressed and I can see a nice band on the agarose elfo.
Thank you.

Hi,
I have problem with my QPCR experiment. I got the same Ct value for the expressed and non-expressed gene. On the agarose elfo there is no band as there is no gene expression. Why I still got the same Ct value as my housekeeping gene. My housekeeping gene highly expressed and I can see a nice band on the agarose elfo.
Thank you.

Hello Erika,

Thanks for your question. I think we would need to have the full details of your assay and the targets before we could identify the issue with this experiment. Please send us an email at techsupport@biosearchtech.com with the probe and primer designs, the intended targets, and an image of the qPCR data demonstrating the issue. We look forward to hearing from you so we can fix this issue with your assay.

I have a question, I've ran numerous experiments and I've inconstantly found my CFU values to drop towards the end of my protocol. I have normal curves that plateau but after about 5-6 cycles in the plateau the CFU values start to decline. Could this be due to something in my reaction mix or a change in UV absorbance in my tubes? Or am I completely off?

Hi,

I am doing qPCR experiments, but i have a query for threshold setting. Do we need adjust the threshold value for each run of the same gene. Also when i perform the reactions with two different genes, my melting curve changes based on the genes i work.

Hello Pavan,

yes you can set the threshold value manually at fluorescence value which will provide a robust crossing point which is typically early in in the exponential (linear portion of curve) amplification phase. This setting can be used across runs of the same assay to determine Ct. Also in a melt curve the character of this derivative curve will vary according to the amplicon assuming the signal generation from this curve is a dsDNA binding dye. This is becuase each amplicon will create its own melt profile and Tm. Please feel free to contact us at LGC Biosearch with any other questions you might have.

Thank you Mr. James for your suggestions. I will try with this. I have one more query with respect to melting curve. i need to share my results with you, so that you can help me in resolving the problems. can you plz reply the mail id to send you my results.

Thanks

Hello,

You can contact us at techsupport@biosearchtech.com to send additional data or questions. We look forward to hearing from you.
-James

Hi: We are testing expression of several genes using SYBR Green chemistry for the first time. We have now tested 21 primer sets and they give nice single bands on regular PCR. Upon qPCR, we get single melt curves with most sets while 3-4 sets gave us either another a minor primer/dimer peak or multiple peaks (only with one set). But when we ran the reactions on the gel, we found that there were primer/dimers in most samples along with a nice band of interest plus another band close to primer/dimers. In one rxn we had two specific bands, but on melt curve analysis we still got a nice tight single peak! We are titering our primers now to see if lowering primers can help. But the question is should we believe the melt curve analysis or the gel picture? We ran NTC controls for each sample and in most cases we did not get any amplification and in the few cases that we did, we got amplification after cycle 34-35. The question is why are we getting nice tight single peaks upon melt curve analysis when we can see multiple bands on EtBr staining? Any comments would be really helpful.

Hi,
I have just started working with SyBr Green qPCR. The problem is that the RFU I get is very low and even after seeing some amplification in Real Time (Ct around 26) and a melt curve with a single peak at 79.5 degrees, the band i observe is not at the correct size. The expected size is 340 bp and I observe between 100-200 bp. 18s however worked well although even that showed lower RFU but band was observed on the gel. Any help would be highly appreciated. Regards

I am running qPCR using SYBR Green. I analyse my data using absolute quantification-2nd derivative max. I am running 40 cycles however the highest Ct value I get is 35. It is never 35.2 or 35.7 etc, just 35. I use the melting curve to decide if this is "real" detection or "noise", but why are the Ct values not going beyond 35 when I am running 40 cycles?

Hello Paige,

The Ct value refers to cycle threshold, or the cycle in a qPCR reaction where the fluorescent signal in the reaction rises above the threshold of detectable signal over that of the basal background fluorescence. In other words, Ct value is determined as the point on the x-axis of a qPCR curve where a replicating sample of target DNA accumulates sufficient fluorescence to cross an arbitrary threshold. Typically, the threshold is adjusted to the mid-point of the exponential phase of the PCR.

And so if the Ct value of Sample A is lower than that of Sample B by 3 cycles (assuming 100% reaction efficiency), then the sample A contained 2^3 = 8 times more template than sample B as the number of template molecules will double with each PCR cycle. Therefore Ct value is not the same as that of the number of total cycles you run. The cycle number refers to the number of times the thermocycler will perform a complete PCR cycle including denaturation, annealing, and elongation. The Ct refers to a determination of the point that the fluorescent signal becomes robustly detectable above the basal fluorescence by the qPCR instrument.

Also, relating to your comment on separating the “real” signal from the “noise” by a melt curve. Please be aware that double stranded DNA binding dyes like SYBR green will bind to all double-stranded DNA produced in the PCR reaction, this is including primer dimers and any non-specifically amplified products. Therefore only looking at the melt curve alone is sometimes not sufficient to determine whether the amplified material in the reaction is the targeted sequence. As such, we recommend a probe based detection method which adds an additional layer of security into the detection of the target amplicon.

Please refer to https://www.biosearchtech.com/support/education/gene-expression-qpcr on our website. The section “Principle of QPCR” explains the Ct values clearly with a figure. This Wikipedia page https://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction#Modeling also contains detailed explanation should you need more detailed information.

Please feel free to contact us at techsupport@biosearchtech.com if you have any other questions. We will certainly be happy to help.

Regards,

Hi,
I am trying to improve the efficiency of a qRT PCR reaction using a Taq-Man probe. I believe that I need to add a carrier nucleic acid to my 1 -step reaction to improve the accuracy between replicates at the low end of my curve. Can you recommend a good carrier molecule to use? I have seen salmon sperm DNA and tRNA. Where would I get such a thing that is PCR-grade? Do you have any other suggestions for improving the Ct standard deviations and efficiency (I have tried different master mix chemistries, titrating MgSO4, primer/probe ratios. My efficiency is right around 60%, my replicate standard deviations are around 0.6, my slope is right around -4.5.
Thanks,
JN

I have a doubt, I am using a primer set which works for two different strains of same virus as the reagion amplified is same.I have used this primer with the samples infected with these viruses previously which has worked for me. Now it's amplifying one strain but not the other.I am using SYBR green and one step qRT PCR.however the samples are getting amplified in normal PCR.I have tried making new primer dilutions,changing standard curve dilutions, increasing cycles of experiment.While increasing cycle there is amplification also in negative samples and water,contamination is not possible I am sure of it. Please help me.

Hello Viji,

It is possible that your sample has mutated and the target sequence is no longer what is expected. This is a big concern with pathogen detection assays targeted to species with a high degree of sequence variation among the different isolates. I would suggest verifying the sequence of the target region in your virus sample or else sourcing a sequence confirmed sample of this virus to confirm this is the case. If you require more troubleshooting please contact techsupport@biosearchtech.com. Have a great day.

I am trying to obtain standard curve and its been more than 10 times I am getting stack peak of all 5 std conc. Also NTC is showing peak. Newly ordered primers and sybr green was used but still Iam facing the same issue. Can you suggest me where to troubleshoot and what is the issue?

Hello Aruna,

This could be caused by a number of different issues. Could you please send us an email at . In the email please include the details of the assay (target sequence + primer designs), the reaction conditions, the dilution series you are using. Please also include images of the non-baseline normalized amplification curves or perhaps just the data file which we might have the software to open. I look forward to hearing from you.

Hello,
I have a question regarding melting peaks. I am testing several sets of new primers and all are producing a specific product with one melting peak and no primer dimers. However, one set of primers produces a melting peak with much lower fluorescence than the others. Is this of concern? I assume this means the primers are not working well, but is there any way I can troubleshoot this with conditions or do I need to design new primers? Thank you.

Hello Molly,

This is hard to determine without seeing the data. If you would like us to review the data please send details of your experiment and images of the amplification and melt curves to . We look forward to hearing from you.

Hello Group,

I am a PhD student of Spain and i have a question about real time PCR. I have a Taqman probe reporter (FAM5l) and quencher (TAMRA3l) and i have a high background in the amplification plots. Its an unussual thing but in some samples or somentimes in the negative control it happens, Is it the probe and primer concentration? or a tecnical problem of the software 7500? Maybe a problem with the threshold or the baseline? Thanks

Hello Jose,

Thank you for your question. Please contact us at Techsupport@biosearchtech.com with the target sequence, the sequence of the probes and primers, and the data file from the ABI 7500. With that information we should be able to troubleshoot this assay. I look forward to hearing from you.

Best,

Jim Flynn

Hello,

I am trying to obtain my standard curve on a RT-PCRq assay. I am working with a RNA virus that I've cloned into plasmid DNA and transcribed it in vitro. The quantification of this RNA using spectrophotometer was 1000ng/μl. From that, I diluted it on 1/10 serial dilution (3 μl of RNA into 27μl of RNasefree water Eppendorf, vortexing and pipetting from that into a new tube, and so on). I chose 5 points (10 ⁸ to 10 ⁴), as recommended for standard curves, to establish the sensitivity of my assay. After this background, here is the issue: Using 2μl/reaction I don´t get any amplification on the last point, 10 ⁴ samples (3 replicates), although the standard curve looks fine in terms of r², efficiency and slope. To solve it, I decided to increase the amount of RNA per reaction to 5μl, which I got amplification at all points, however the curve parameters were all bad. I already repeated this last test (using 5 μl/per reaction) and got the same problem. I have no idea what is going on my assay. I already checked on conventional RT-PCR the annealing performance on my primers set, and it´s all good. Also, ran a RNA gel to check the quality of my RNA, and it’s good.
I currently use the QIAGEN QuantiTect Probe RT-PCR on 25 μl at final volume and Applied Biosystems 7500 Instrument.

Hello Tamisa,

Thank you for your question.

To help us better understand and assist you with the issue, could you supply us with details of the assay? Please provide us with your primer and probe sequences, the calculation of your RNA quantification, and the original qPCR data files from the ABI 7500 machine.

Please send these information to techsupport@biosearchtech.com. We look forward to hearing from you and assist you with the assay.


Regards,
Hsiao Yuan Chen

Hello,
Please could you help me to understand the issue I have with my human residual DNA quantitation assay?
I'm using a commercial TaqMan assay which is provided with a genomic DNA standard. When I use this standard to spike my sample, I have good recovery and good % CV. However, I tried a genomic DNA that we have extracted ourselves and with this recovery is very low and % CV abberantly high. When I test a standard curve in PBS with this DNA we have prepared, I obtain a good curve with good R² and qPCR efficiency. These results are reproducible accross different experiments and users.
Would you have an idea, why my results are abberant when I use our prepared DNA?

Hello Bea,

Sorry to hear that your assay is giving you some trouble. I assume your are to a Human Residual DNA kit provided through another vendor as we do not make one ourselves. Unfortunately we can not offer technical guidance on product from another supplier, but perhaps provide some suggestions about the cause of this issue. The standard provided in the kit is likely a double stranded synthetic template. These will tend to amplify very robustly within an assay. Extracted human DNA will typically be far more complex mixture which will perhaps not have the same sort of consistent amplification which you observe with the synthetic standard. We do not know the composition of the assay so it would be difficult to provide any additional details on how to proceed. I would recommend contacting the manufacturer of the kit for explanation of the performance of the sample. They might have some suggestions on how to improve assay performance.

Thanks Flynn,
Indeed it's possible that the commercial standard is more robust.
I'll investigate further through the vendor.
Thanks a lot so far!

Are there any online tools to analyze a group of primers at the same time for possible primer-primer interactions? I'm getting some non-specific signal on my assay, and want to determine if any of my oligos are interacting with each other. Thanks

Hello Mark,

Thank you for your question. Unfortunately, we are unaware of an online tool with the stated function.
Please let us know if we could assist you with any other issue.

Regards,

Hello there,
I did run a qPCR for 18SrRNA gene with different cDNA concentrations (in ng of initial RNA) such as 25, 50, 75, 100 and got the more or less similar Ct ~27-26 to all of these concentrations. previously I did try lower concentrations 2, 4, 6, and 8 ng's in that case the Ct appeared at ~30-31 cycles. Not sure what is going wrong. For 18SrRNA Ct is expected to appear much earlier in PCR. I also run three other genes using 25, 50, 75 and 100 ng of cDNA and all these genes have the similar problem, again ~similar Ct's in all different concentrations. What could be the possible reason? Will you please help me to trouble shoot? Thank you

Hello Suhas,

Thank you for your question. To help us better understand and assist you with your assay, could you supply us with details of the assay? Please provide us with your experimental protocols, the sequences of your primers, probes and targets, and screenshots of your qPCR data (not baseline-subtracted) or the original data files from the qPCR instrument.

Please send these information to techsupport@biosearchtech.com. We look forward to hearing from you and assist you with the assay.

Regards,

Hello there,
I have one question. I am currently working in an absolute quantification. I have a synthetic standard of 150 pairs of bases approximately. I have made 1:10 dilutions with the same standard but when I see the results displayed on the equipment, the CT are exactly the same or just with a little variation, they don’t achieve 3.3 cycles of difference between one solution and other. I have made sure to do the solutions properly. Furthermore, I have tried with a positive sample, with this one, everything results perfect but not with the standard. Can you please help me to understand what can be happening?

Hello there,
I have one question. I am currently working in an absolute quantification. I have a synthetic standard of 150 pairs of bases approximately. I have made 1:10 dilutions with the same standard but when I see the results displayed on the equipment, the CT are exactly the same or just with a little variation, they don’t achieve 3.3 cycles of difference between one solution and other. I have made sure to do the solutions properly. Furthermore, I have tried with a positive sample, with this one, everything results perfect but not with the standard. Can you please help me to understand what can be happening?

Hello Luz,

Thank you for posting your question. To help us better understand and assist you with your assay, please supply us with details of your assay. Please provide us with your experimental protocols, the sequences of your primers, probes and targets, and screenshots of your qPCR data (not baseline-subtracted) or the original data files from the qPCR instrument. Please also let us know the template concentrations for both the synthetic standard and the positive sample.

Please send these information to techsupport@biosearchtech.com. We look forward to hearing from you and assist you with the assay.

Hi,
I am observing early CT in the control, expected CT is 28 and system is showing it as 20.25. Please update me the possible causes.

Hello Abhay,

Please email technical support at techsupport@biosearchtech.com with the full details of your experiment and data demonstrating the issue. If possible, please also include the sales order number for the oligos related to this assay. We look forward to hearing from you.

Sincerely,
James

Hi there, quick question. I tried to set up for the first time a 96-well plate real time machine. I run the full plate with approximately half the wells empty. I got Ct values for all the wells (even the empty ones). My samples gave the expected result but all the empty wells also gave very close ct values (27 vs 30). Is this a contamination problem? What should I do?

Hello Katerina,

We are sorry to hear you are having issues with your assay. Please provide us with the full details of your experiment setup, and screenshots of your qPCR data (not baseline-subtracted) demonstrating the issue. We have the software for some common qPCR instruments so you could also try sending us the original data files from your instrument. Please send all the details and data to techsupport@bioserachtech.com. We look forward to hearing from you and assist you with your assay.

Regards,

Hello,
I observed that amplification curve shows Cq value at around 23 cycle but in data it shows N/A. I could not understand this problem. Thank you

Hello Kiran,

We are sorry to hear that you are having some issues with your data. To help us better understand the issue and assist you, please supply us with the full details of the assay. Please include your experiment setup and screenshots of your qPCR data (both non-normalized and baseline-subtracted) demonstrating the issue. It may also be helpful if you could send us the original data files from your instrument as well. Please send all the details and data to techsupport@bioserachtech.com. We look forward to hearing from you.

Regards,

I did qrtpcr but I am getting always two peaks I diluted the primers also but I am not able to get single peak Tm temperature of primer is ok I dont know where I am doing mistake. Can you please suggest me.

Hi Sangeeta,

Thank you for leaving a comment. Please supply us with the full details of the assay. Please include your experiment setup and screenshots of your qPCR data (both non-normalized and baseline-subtracted) demonstrating the issue, as well as your target sequence and probe/primers sequences. It may also be helpful if you could send us the original data files from your instrument as well. Please send all the details and data to techsupport@bioserachtech.com. We look forward to hearing from you.

Regards,

Correction: Please send all the data to techsupport@biosearchtech.com

Hi guys

I am currently undertaking TaqMan SNP genotyping with FAM and VIC labelled probes. My SNP has an MAF of more than 0.5 meaning that the minor allele is expected. It is also close to data sequenced among the Luhyia of Webuye Kenya which show distribution of the three genotypes at varying levels. However, my results are turning out heterozygous for every sample. I have genotyped more than 150 samples, tried changing annealing temperature to 62 or time to 45 seconds but the results are still the same. The labelled amplification is starting earlier than VIC though all amplifications begin from around the 20th cycle. Is something wrong with my assay? Please assist.

Hello Owuor,

Thank you for your comment. The assay described in your post would indicate that this is an assay format supplied from Thermo Fisher and not through LGC Biosearch Technologies. I would suggest contacting their technical support team to resolve this issue.

As an alternative, we would be happy to take a look at the existing design and work with you to convert this to our BHQplus assay format which is ideal for SNP allele discrimination. If you would like us to redesign the assay simply provide the target SNP accession number and the complete information for the primers and probes. I look forward to hearing from you. Have a great day.

Hello! I'm trying to validate my primers with serial cDNA dilutions and in some cases I have a slope bigger than 4, and R2 of 0.99, what means that my primers have low efficiency (around 75-80%). There's no primers dimers occurring since there's no amplification on the negative control. The Tm difference between the primers is lower than 5 degrees... I was thinking about change the primers concentration, do you think it could increase the efficiency? Please, help me!

Hello Alice,

Thanks for your question. We will reply directly to you from our technical services team. this sounds like a case where design or reaction conditions might require some optimization. Our team will contact you shortly. Have a great day!

Hi, I isolated RNA from specific cell type resulting in less RNA but i could synthesize cDNA from that. I ran qPCR and i get good amplifications with my reference gene GAPDH but i couldn't get good amplification in few wells for my gene of interest. We have validated the primers and have been using it for a while. It works well in 96 well plates. Since i used 384 this time, for easy pipetting, i aliquot the master mix along with primers and cDNA for a sample together in a tube and from that tube, i pipette into three wells in a plate for triplicate. Is there a possibility that the cDNA doesn't mix well and due to that, i couldn't see any amplifications?

Thanks
Richard

Hello Richard,

We have responded to your question to your email address. Please don't hesitate to contact us at techsupport@biosearchtech.com if you have any additional question.

Regards,
Hsiao Yuan

Hello everyone,

I'm doing tons of qPCR for gene expression analysis (absolute quantification).
My problem is: a standard dilution series (from 10^7 to 10^2), for a specific gene show strange multiple peaks in the melt curve.
to be clear I'll describe it

10^7 -> one peak @ 84.5 °C
10^6 -> one peak @ 84.5 °C with a little bulge on the left
10^5 -> two peaks one @ 82.3 °C and one @ 84.5 °C
10^4 -> one peak @ 82.2 °C with a little bulge on the right
10^3 -> one peak @ 82.2 °C with a almost invisible bulge on the right
10^2 -> one peak @ 82.2 °C

all the sample tested have a concentration below 10^4 and they all show a peak @ 82.2 °C

I made the plate two times:
the first time I thought I made a mistake.
For the second I have been really careful but the result was the same.

I surfed the internet but i didn't find any explanation about this.

However for the standards the amplification curves were perfect.

So my questions are:
- do you have any idea regarding the two peaks?
- do you think I can use these results?


ThankYou

Cheers

Matteo

Hello Matteo,

Apologies for our delayed response and thank you for your question. Please check your email to which we have sent the reply. Have a great day.

I am running qPCR for quantification of DNA. I have two questions:
1. i have a good standard curve, but when i run the reaction including the standards with the unknown samples, the efficiency changes, it decreases.
what can i do about this.
2. some of my unknown samples amplified at earlier cycles than any of the standards, is this wrong?

Thank you for your question. In order for us to better assist with your issue, please supply us with the Full Details of the assay. Please also include your experiment setup and screenshots of your qPCR data (non-normalized/not baseline-subtracted) demonstrating the issue, as well as your target sequence and probe/primers sequences. It may also be helpful if you could send us the original data files from your instrument as well. Please send all the details and data to techsupport@bioserachtech.com. We look forward to hearing from you. Have a great day.

Hi I am tired to running qpcr.My aim is to clean negative target but I have totally 52 sample I had clear negative control for first 32sample but later when I try to run rest of the sample every time I am getting amplification in negative control what could be possible reason for the amplification of the negative control

Thank you for your question. In order for us to better assist with your issue, please supply us with the Full Details of the assay. Please also include your experiment setup and screenshots of your qPCR data (non-normalized/not baseline-subtracted) demonstrating the issue, as well as your target sequence and probe/primers sequences. It may also be helpful if you could send us the original data files from your instrument as well. Please send all the details and data to techsupport@bioserachtech.com. We look forward to hearing from you. Have a great day.

I am doing plasmid dilutions in water, and we can't seem to get consistency between duplicate dilutions run in triplicate. I have heard about linerarising, but when diluting is there any rules about how long to mix for, or how long before doing the next dilution?

Hello C E,

Thank you for your questions. One of the possibilities may be the preparation of the dilutions. Were you using any carrier DNA in the preparation of the dilution series? Our speculation is that you might be observing the drop-out effect when doing dilutions of limited content of target DNA. This could be that the template did not make it into the reaction at the concentrations you thought it was due to the DNA being absorbed into the plastic surface. Plastics are porous surfaces and well known to absorb some portion of their contents, whether proteins, DNA, or other biomolecules. When you may be manipulating only one or a few molecules of the target sequence the drop-out becomes much more apparent. For that situation, we recommend performing all dilutions with a buffer containing nucleic acid unrelated to the target, the "carrier", and we frequently use yeast tRNA. This carrier molecule is used in vast excess, e.g. 100 ng/uL, and so preconditions the plastic so that the target molecules are less likely to become absorbed. We think this may potentially explain and solve the issue you are having.

Something else to check could include making sure the pipettes are properly calibrated; vortexing and spinning down before proceeding with the next dilutions. These seems obvious but it wouldn’t hurt to be extra cautious. I hope this helps.

Happy Holidays!

how to differentiate between positive and negative sample in qPCR. If the curve is exponential at 100 relative flourescence would that be positive. Is there correlation between relative flou. and sample being + or -

Hello Ahmed,

Thank you for the question. For qPCR, only the end users can determine what their own experimentally derived threshold level is, so they can discriminate between true low level RFU versus signal noise. Make sure the qPCR instrument is calibrated for the dyes being used and check the manufacturer's specifications for their recommended peak height minimum RFU, anything below which is judged very cautiously.

You can view more information on calibration and our calibration dyes via the following links:
https://www.biosearchtech.com/support/education/fluorophores-and-quenchers/qpcr-dye-calibration-standards

https://www.biosearchtech.com/products/qpcr-and-snp-genotyping/dye-calibration-standards

Regards,

Hi
We having a huge problem with qpcr in my lab. We are currently working on miRNAs. We started off by doing a normal PCR to check the cDNA and there was amplification. There wasn't anything in our negative controls so we know that we don't have any contamination in our samples.

The problem occurs when we transition from conventional PCR to qPCR. We don't have any amplification in wells containing our template. However there is still no amplification in our negative controls which is expected.

We then checked our primers on a gel and it was fine. We suspect though that the concentration of the primers are not consistent as some people in the lab don't have any product on the gels while other bands are very faint and others intensified. Furthermore, we tried to increase the concentration of the cDNA in a conventional PCR but now it seems like the reaction is being inhibited (There was only amplification for one of our miRNAs where we previously saw amplification for each of our miRNAs).

Do you have any ideas on what we can try next?

Please help!!!

Hello Sharneal,

Thanks for your questions. Since you mentioned that you got amplification from conventional PCR but not in qPCR, the issue might lies in the qPCR process. It would be very helpful to better understand the issue if you could provide us with your detailed experimental procedures, data, sequences, qPCR device and the fluorophore used, and screenshots of the non-normalized (not baseline subtracted) qPCR data, as well as the Sales Order number or the Sequence Set number of your probes and primers. Please email all this information to techsupport@biosearchtech.com.

It is good to know that your negative controls results stay negative. It might also be helpful to describe in your email how the positive and negative controls were set up and how the positive controls performed.

Also, to check whether the PCR reaction was inhibited due to the reverse transcription process, you might consider using synthetic oligos bearing the sequences of your target cDNA as the template, and compare the PCR or qPCR results of reactions using synthetic or reverse transcribed template. Synthetic oligos can be ordered via our Custom Oligos program on this page: https://www.biosearchtech.com/products/custom-oligos/custom-oligos#?tab=product-info

We look forward to receiving your email.

Regards,

Hello-with one of my Cy5-BHQ2 probes, I observe a baseline drift tha is asymptotically approaching a plateau across 40 cycles. It never reaches plateau. IS this due to secondary structure in the probe that is SLOOOWLY unfolding or?

Hi Brian, Thank you for reaching out. Our technical support team would be more than happy to answer your query! Could you please send over the following to techsupport@biosearchtech.com?
-Raw non normalized data
-Sales Order number
-details about your experimental set up
-the qPCR instrument you're using
We're looking forward to getting in touch with you.
Best,
Ashley

Hi, I was surfing the internet and reading a lot of articles, and I encountered this discussion, I was wondering if I did a qPCR, but I didn't set a proper baseline or threshold, would it affect the results of higher Ct value for the replicated genes??

Hello Hadeel,

Thank you for your question. I am not sure that I understand your question completely, but let me try to answer it. The Ct value could be affected by many factors, such as the master mix used, assay design, reaction efficiency, and the reporter dye used, just to name a few.

It is important to set the threshold correctly, and more appropriate to manually select the threshold value. The qPCR software will often pick an inappropriate value which will result in spurious results (for example, assign a Ct to a NTC which is coming up very late in the assay).

It is also more appropriate to use the same threshold across repeated runs of the same assay, and to do this it is necessary to assign the threshold manually. If you let the instrument's software automatically choose the threshold every time then it will assign a different threshold every time, because the software does not know that you are running the same assay repeatedly, and it will likely introduce variance from run to run.

If the threshold is set differently or incorrectly, then the Ct values would likely be affected for all assays across the board, and may not be limited to assays run with lower amount of template.

For more information regarding qPCR, you could visit our qPCR design lab’s reference page (http://qpcrdesign.com/qpcr-library) or this FAQ page on our website (https://www.biosearchtech.com/support/faqs/fluorogenic-probe-and-primers/i-am-a-beginner-at-realtime-qpcr-does-biosearch-technologies-have-information-which-will-help-me-to-design-my-assay) I hope this helps.

Regards,

Thank you so much for your reply, I really appreciate it, you understood me perfectly, I was wondering have you ever tried miscript PCR array, since I have a problem with this technique and I need to know what's wrong with it.
Thank you..

Hello Hadeel,

You are very welcome. Unfortunately we do not have experience with the miScript miRNA PCR Arrays product, and so you might consider contacting the manufacturer for more guidance.

Regards,

If I have amplification just with 38 cycles, but the point is outside the standart curve. Can I use this value? Is it could works to diagnosis insted quantitative assay? Is correct put the threshold up to fix it?
PS: negative control is ok.

Thank you
my very best

Hello Gabriel,

We would advise against using any data point falling outside of the standard curve. Standard curve should be performed with varying concentrations of the DNA template with each probe and primer set used. The results of the dilution series and standard curve will give you an idea of the limit of detection of the reaction. It could be possible that there are too few copies of the template in the reaction, resulting in the late Ct values of your samples.

Regards,

Hello
i have low primer efficiency so could you please look at the picture of standard cure and let me know whats the problem.
thanks

Hello Esmaeil,

You can send screenshots of your qPCR curves in non-normalized (not baseline subtracted) form to techsupport@biosearchtech.com. Please also include a detail description of your experimental setup, protocol, as well as the Biosearch Sales Orders number of the primers and your target and probe/primers sequences.

Regards,

What does it mean to have a high Ct values above 32, and at the same time the curve looks shifted to the left, most of the products detected at late cycles??

Thanks.

Hello again Hadeel,
In order for us to understand the issue, please send screenshots of your qPCR curve in non-normalized format (not baseline subtracted) demonstrating the issue, as well as the Sales Order number of your probes and primers, a detail description of your experimental setup, and protocol. Please email all the information stated above to techsupport@biosearchtech.com. Have a great weekend.

Regards,

Hi,
I am doing some gene expression analysis by SYBR Green Real Time PCR and in my experiment, I am using beta actin as the house keeping gene. Now for some particular genes I got weird results in triplicate. For example, in one well the Ct value is 31 but in the other two wells the values were "undetermined", but the beta actin values are always good in triplicates. I am confident with my pipetting because for other genes and for the beta actin gene I never ever had that kind of weird result. A very efficient and experienced technician in our lab tried with that particular gene of that particular gene and he also got the same type of result, i.e., in one well the Ct value was like 30-31 but in other two they were "undetermined". We repeated several times the experiment but always faced the same problem. Can anyone help us to fix the problem please?

Thanks
Priyanka

Priyanka,

have you considered the possibility that perhaps your cDNA/genetic material is in an interface that is highly non-homogenous. Genomic dna is very complex and not a suspension so you could have arbitrary results as you describe. So, no it might not be YOUR pipetting as you said, but it could be pipetting in general. Have you changed the location of where you put those samples in the 96-well plate reader (assuming it's strategene)?

gluck.

Hi Payal,
Thanks for your response. I totally agree with you. Yes, the genomic DNA could be non-homogeneous and if the gene of interest is lowly expressed, then definitely it could be a possibility that one may have detectable Ct value in one well while undetermined in other triplicate. Now to answer your query, I am saying that I already changed the location of the sample in the 96 well plate but still the problem remained.
So, to get rid of this kind of problem, what is your suggestion?
Thanks in advance.
Regards,
Priyanka

I have a question regarding my q pcr results, as the temperature increases fluorescence should gradually decrease in the melting curve, but when im using cDNA it increases with temperature from 65-70 C, then suddenly decreases from 70-75 C, but it is not happening when im amplifying same pcr product for DNA, can anyone suggest a reason for that?

Hello Udee,

We are not exactly sure what your problem is based on your description. Are you concerned that fluorescence is dropping off too rapidly in the melt of product from cDNA template? Or that you do not have a product in the gDNA reaction? In a typical gene expression experiment, one wouldn’t want to amplify anything from genomic DNA with primers designed to target a specific transcript (cDNA), so you should not have a product (and thus nothing to melt) when amplifying from genomic DNA.

We have experts here at LGC Biosearch Technologies ready to help you troubleshoot your qPCR experiment. If you can send us your melt curve data images along with assay condition/design, we can take a look and help solve your problem. Please send the data/information to Pet.techsupport@LGCGroup.com.

Regards,
Hsiao Yuan

Hello. I'm using SYBER green realtime pcr to detect some genes expression in a cell line, A2780. The problem is that I get very low or undetermined Ct values for most of the genes, even some housekeeping ones. But b-actin has a normal Ct value. I've tested reaction condition and primer sets with other cell lines and I'm sure about their accuracy, but I'm totally confused about this one! Could there be some changes needed for this?

I'm so sorry I meant high Ct values and most of the time undetermined results

Hello Bahar,
Apologies for our delayed response. It sounds like you have poor quality or insufficient amount of cDNA— the lack of amplification for housekeeping genes suggests it is a DNA issue. If B-actin works (at approximately the same Ct value as B-actin works in another cell line, where the assays in question also amplify) it could mean that your genes of interest are not expressed in that cell line.

What does the Ct value of B-actin look like in this problematic experiment, vs. an experiment/different cell line where the assays worked? A few cycles shift may mean you are still able to detect B-actin easily, but the other targets are so low that they are beyond the limit of detection. We hope this helps.

Regards,
Hsiao Yuan

Dear Hsiao
Thanks another for your reply. For other cell lines B-actin Ct values are in range of 13-15, and for this specific cell line it's 13.
Are you suggesting that cDNA increase may help solve the problem?

Hi,
I am using a MycoTOOL PCR detection kit from Roche in order to detect Mycoplasma in different samples. I am using it as an ON/OFF read-out, no quantification needed.
In a way of saving money ($5,000 for the whole kit), I just tried to reduce the amount of the reaction mix which is 50 uL final (with 30 uL of master mix and 20 uL of template) to 25 uL final (with 15 uL of master mix and 10 uL of template).
I got pretty much the same Ct values for the positive control but interestingly the slopes of my amplification curves were decreased massively.
I sounds like the efficiency of reaction decresed, but I kept the same mix/template ratio so I am wondering why?

Thanks!

Hello Jonathan,

To better understand the situation of your specific assay, could you please provide us with all of the following information?
Please provide us with:
1. The Biosearch sales order number or the reference number of the primers and probes used in the assay in question.
2. Screenshots of your qPCR amplification data with No baseline subtraction demonstrating the issue.
3. A detailed description of your experimental setup, condition, and protocol for the reactions of both volumes.
4. Other data demonstrating the issue, such as the slopes of your standard curves of different reaction volumes.

Please send all the items requested above to techsupport@biosearchtech.com. We look forward to hearing from you.

Hello
I write because I would like to know how look like one degraded curve standards?,
I was working with my curve standards. later of two months I try to process and my curve standards present a particular distribution. there aren't curve and some dilution does not show amplification.
I prepared again curve but this was not useful.
I sterilised all material.
I don't know what do?

Hello Helena,

It sounds like you would like to know what the amplification curves would look like if there are degradations occurred in the reaction. Without seeing your data we cannot speculate what kind of issue you encountered, however we can offer the following thoughts for your consideration.

DNAse contamination (for example, due to bacterial contamination of reagents) or reducing agents carried over to the reaction would look like progressively increasing baseline fluorescence, similar to the first and last diagrams shown in this blog article. Traces amounts of microbial contamination in reagents can be enough to introduce the DNAse, as a microbial cell that makes it into the reaction would be lysed at the high temperature for PCR, releasing lots of DNAse, which degrades the probe and releasing the fluorophore from the quencher. Reducing agents such as DTT sometimes present in master mixes (and different batches of polymerase will have different levels of DTT), or might be carried over from reverse transcription reactions. All dark quenchers, including DABCYL, our Black Hole Quenchers, as well as the Iowa Black and Zen quenchers from IDT contain an azo bond (N=N) that represents a site of potential reduction. Once the azo bond is reduced this can render the quencher non-functional.

For the dilutions that didn’t show amplification signals, you might run the reaction product on a gel to check whether the issue lies in failure of the PCR reaction, or failure of signaling of the amplicons.

Best,

I have a problem with respect to the melt curves in the recent times. Previously I used to get very good curves, but since a few experiments even though using all the same chemicals, I donot see any background noise that you usually see in the melt curve. Previously the baseline used to be set at 0.2 and analysed and I get good results. But now I have to alter the threshold a lot. I donot know why this is happening. I used as positive controls, the samples which gave good results before, but for this sample as well I donot get any background noise and good threshold. can you please let me know could be the problem?thanks in advance

just more information for my previous question, I use TaqMan probes and lowrox, and in my first experiment even I do a duplex PCR, I had very good results and now even with individual genes I donot have good melting curves

Great post, very informative. Thank you for the insight.
Request you to have a look at an article based on fermentation development and scale-up - by Eppendorf.
Link: http://promotions.pharmafocusasia.com/fermentation-development-and-scale-up

I perform qPCR with SYBR green, GAPDH control to test my skill. Samples are liver, brown fat and draining lymph node. The liver sample is amplified well (Ct value is 17-18), but brown fat and draining lymph node are not amplified (I tried more times). I used 5ug RNA for cDNA synthesis, then dilute cDNA 5 times to run qPCR in 384well-disk. Can you give me any advices for addressing this problem.

cDNA synthesis: 5ug RNA, 1ul dNTP and 1ul oligo dT for 1st program and then (4ul 5X FS buffer, 1ul DTT, 1ul RNase OUT, 1ul SS III Transcriptase) for 2nd program
qPCR 8ul Mix (2ul D.W, 5ul SYBR green Master MIX and 0.5 each F&R primer) and 2ul cDNA (5 times dilution)

Hello,

I'm preforming qPCR with SYBR, testing expression of several different genes in several tissues. cDNA in wells is diluted 100 times from reverse transcription mix (starting with 1 ug of RNA). With some genes, I get great disparities among technical replicates. I think poor pipetting / mixing can be excluded as a reason for that since with other genes, I get a perfect match between technicals, experiments are run back to back, cDNA is taken from literally the same tube. Poor primer design can also be excluded: good results with the same primer in one tissue, bad in other and primers were purchased from a third party.

are low copy number to blame? would increasing amount of cDNA in wells resolve the issue? or it is poor pipetting/mixing after all?

Hello,
I have problem with my amplification plot in multiplex real time (FAM, HEX and Texas red). Most of my specimen curves do not remain flat in the plateau phase.
could you please help me through that?

It would be best to raise a case with our technical support team as there may be a lot of reasons why you're seeing this. If you go to https://www.biosearchtech.com/about-us/locations you'll be able to submit a support request. You can also check out this link for more information: https://www.biosearchtech.com/support/education/multiplex-qpcr

Hope this helps!

My positive control gives way lower RFU than the QC report from the manufacturer. This had been repeated for multiple times. May I know why?

any

Hi!

I am currently performing qPCR analyses of the RNA (or rather cDNA) of endothelial cells that were previously stimulated. I have different primer pairs for different targets. I have done these analyses 1h/3h/6h post-stimulation of the cells, unfortunately some primer pairs don't work for all of these time points (the melting curves look odd). So my question is WHY do some primer pairs only work for some but not all time points? I am analyzing the expression of endothelial adhesion molecules.

Thanks so much in advance!

Dear Larissa,

Many thanks for contacting Biosearch Technologies with your technical query, and providing details of the issues you are experiencing. There are several points to consider which may explain the results you are observing.

Firstly, it is important to include positive controls for each set of primer pairs at each of your time points. We would recommend an RNA positive control, as this will ensure that both your reverse transcription and PCR reactions are performing optimally each time.

Secondly, it is important to ensure that your reverse transcription and PCR reagents are being stored and handled correctly. For example, if you are making a "bulk" master mix for all your time points (to reduce variability), your reagents may be decreasing in stability during your time course, which would result in variable results.

Finally, there could be a change in expression of your targeted isoform after stimulation or abundance of the target at later time points. There may be a biological change in expression or inherent enzymes (e.g. RNases) present in your sample which may be impacting the integrity of your target of interest. Considering the inclusion of isoform inclusive primers for your target and RNase inhibitors may be a option.

I hope our advice is informative, and we wish you luck in your future experiments.

- Scientific support team