As usual a lot of critical info is missing in the question: which system are you using for qPCR: SYBR-green, TAQMAN ... , what is your template, gDNA, mRNAs, miRNAs, lncRNAs, are you expecting a yes/no answer, absolute quantification or relative quantification from your qPCR ... What negative controls are you using? NTC only, or -RT or in the case of gDNA, gDNA from an unrelated species? How do your melt curves look like and how are the CTs of your technical replicates... All this info would help to give more specific answers.

Theoretically, if your negative controls don´t produce any product, and if your technical replicates are consistent (less than 5% deviation, depends on the accuracy of your pipette), the melt curves give a clean and single peak, you would be able to say that the product is not an artefact. You could even sequence it to confirm specificity. However, what will be more important: you´ll need some functional assay to test whether whatever you were checking has a biological effect. It´ll not matter how big or statistically significant differences are, unless a biological function can be shown.

One thing about negative controls: NTC is a bit of a cheater. It only accounts for primer dimers and contamination of your reagents with target DNA/cDNA, so it should of course be included. However, if you work with cDNA, -RT controls are even more important as they´ll allow you to judge whether there are traces of gDNA left that produce either specific or spurious PCR products. Even with intron-spanning primers, you might get some spurious PCR products, so having the "right" size is not absolute prove.

If u r using binding dye and suspecting the formation of primer dimer (observed in most of the cases), it is the convenient point to get Ct at around 34, 35 and beyond--no surprise in that. A melt curve analysis resolves the issue and gives u a clear idea on taking decisions, and gel observation of products supports this (observation of non specificity at molecular weight level, primer-dimer). You can easily conclude!

If it is nuclease activity based setup, u can suspect mild contamination on Ct  below 35 (by comparing with NTC),

but even taqman gives u later Ct at around 37, 38 or beyond. Most of the cases u can simply neglect them!

But acceptable range always depends on the primer and template (abundance range of target on template) system. If ur aim is to interrogate very low level targets, please seek help from ur guide.

Good luck!

Dear Mansi Srivastava

I agree with @Lakshmi Narayana Lavu

But normally the cut off value for Ct is 35, and you should also perform melt curve that will rule out the amplification of your product or primer dimer.

Ct above 35 is not considerable. You need to standardize your PCR conditions. 

Dear Mansi Srivastava!

I used to perform a lot of RT-PCRs as a PhD student. Because in case of a given sample the Ct value could also increase due to a lower expression level and not due to unspecific amplification (which could give false positive results) I tried to exclude as few results as possible. In my PhD thesis I excluded only those results where Ct values were higher than 40 in case of ALL samples. 

Of course, I verified that the melting temperature has only one and identical peaks in case of ALL samples. If the previously mentioned statement is true, than I could trust that no unspecific amplification happened during the reaction.

I am currently preparing a tender which will be based on a lot of RT-PCRs. If anyone has a better, more accurate method or some advice related to this question, it would be very helpful.

Sincerely,

Antal Otilia Tamara

You confirm the ct value 35 or not by checking melt cure to ensure that there should not be any non-specific amplification or primer dimer. If that is clear then it will be acceptable by comparing it with reading in other samples otherwise reaction requires optimization either by increasing templated or primer concentration or quality of cDNA/RNA.

depends if your no template control has any amplification or not. typically real signal is considered to be average ct for NTC - 2. not delta ct ... remember you're concerned with right censoring.

Hi Mansi,

Let's pretend for the moment that we are dealing with detecting a deadly pathogen. Let us also assume that you have proven by melt curve analysis or by direct sequencing that one of your samples with a Cq >35 indeed shows that your amplified product is indeed the pathogen target sequence. Reactions with a starting single copy of target sequence have been reported to appear anywhere from Cq of 35 to 38 in highly efficient qPCR reactions, whereas a Cq of 48 (proving to be a real target amplification) in a 70% efficient qPCReaction is entirely possible (and has been demonstrated). So Otilia and Sachin's comments above are very informative in this regard.

In rough language, a Cq range of ~14 to 38 is most useful in qPCR. But, if primer-dimers and non-specific products start showing up at, say, a Cq of 28, the qPCR starts to be questionable at a Cq of >=28. Like the others have mentioned above, if you have unequivocal proof that your high Cq values are indeed amplifications of your specific intended target sequence, then, they are real and useful measurements. Proof of fidelity for target in reactions yielding high Cq values is a must.  Also, if indeed a Cq of 48 (in a poorly efficient qPCReaction) proved to be the target sequence for, e.g., Ebola, one can make a strong case for not throwing the baby out with the bath water as it were.  Proof by direct sequencing or good melt curve analysis of product is a must in these situations.

Hello,

In my experience, human identification, samples with CT values above 35 have not enough DNA to reliable profiles. Low copy number, non-specific, amplification is found.

I would not believe the real-time result over 35 cycles, as even the negative control will show the amplification result. Or you can choose an alternative method as Taqman probe, which is a more sensitive detection method.

You may accept Ct above 35 if it is probe specific target, means that your negative controls always negative at even at 40th cycle and your amplification is relatively stable and consistent.

There are times that we need to detect down regulation of low expressed genes, or simply low copy of the materials, as long as all controls consistent, and reference genes also in the comparable range i.e Ct not lower than 28 etc.

In order to accept the high Ct value (low expression or low template presence), there is much more requirements for your real time PCR bench practice, such as contamination prevention, such as to use low expression reference genes, such as need more verification across different specimens etc.

Yes, if you have good control.

In routine clinical EGFR mutation test using Qiagen EGFR RGQ test (FDA approval kit), EGFR mutation signal come up after 35 cycle will still be judged as positive as long as  your positive ,  negative control and your signal  fit required criteria.   

As usual a lot of critical info is missing in the question: which system are you using for qPCR: SYBR-green, TAQMAN ... , what is your template, gDNA, mRNAs, miRNAs, lncRNAs, are you expecting a yes/no answer, absolute quantification or relative quantification from your qPCR ... What negative controls are you using? NTC only, or -RT or in the case of gDNA, gDNA from an unrelated species? How do your melt curves look like and how are the CTs of your technical replicates... All this info would help to give more specific answers.

Theoretically, if your negative controls don´t produce any product, and if your technical replicates are consistent (less than 5% deviation, depends on the accuracy of your pipette), the melt curves give a clean and single peak, you would be able to say that the product is not an artefact. You could even sequence it to confirm specificity. However, what will be more important: you´ll need some functional assay to test whether whatever you were checking has a biological effect. It´ll not matter how big or statistically significant differences are, unless a biological function can be shown.

One thing about negative controls: NTC is a bit of a cheater. It only accounts for primer dimers and contamination of your reagents with target DNA/cDNA, so it should of course be included. However, if you work with cDNA, -RT controls are even more important as they´ll allow you to judge whether there are traces of gDNA left that produce either specific or spurious PCR products. Even with intron-spanning primers, you might get some spurious PCR products, so having the "right" size is not absolute prove.

Hello. That depends on your house keeping gene CT. then you can decided about CT35: is it good or not. You can increase the amount of template (c DNA) to challenge this problem.

While trying to detect very low copy number of target gene, it might be necessary to increase the cDNA amount in such a proportion that your loading control gene (which is fairly abundant) CT value lies in the acceptable range. If the CT of house keeping gene is too low, it is necessary to look for a less abundant gene in your system. Otherwise the comparison is biased.

I think that CT values exceeds 39 would not considered target amplicon unless you used a probe for more specificity.

Hello,

I have considered in my research a Ct value below 35 as acceptable for my samples once I checked the melt values to ensure there are not any non-specific amplification or primer dimer. I need to justify this point with a bibliographic cite , sine the reviewers ask me . Does anybody know any bibliographic cite for this purpose?. Thank you so much

Hi Christoph Metzendorf , I was trying to plot a standard curve using synthetic oligo microRNAs. As no one in our lab has worked on this before, I have to start from scratch here. I'm using kits from BioRad for both cDNA synthesis as well as qPCR. I used miRNA concentrations of 10fM, 100fM, 10pM and 100pM for the cDNA synthesis (reaction volume of 20µL) and then took 2µL of the RT product in 20µL of total qPCR reaction. But I only get any amplification after nearly 40 cycles, although this trend is consistent and the NTC shows no amplification even at this number. But I think such high Ct values are not acceptable for synthetic mimetics, so could you please suggest where I could be going wrong?

Arti Tyagi - I have no experience with qPCR of miRNAs and the chemistry of synthetic miRNAs. I can only speculate about possible reasons. You say you get amplification and CTs seem consistent with dilutions; negative controls (-RT and NTC) are not resulting in any kind of product., right?

This means that its working in principle, but something may not be efficient enough. At least 0.1 nM of a specific template sounds enough to me to produce products before cycle 40...but I might be mistaken. You'd have to check in the literature if others have amplified miRNA dilution series and what concentrations were used and which CTs resulted from this. If your CTs are indeed too low there are plenty of possible reasons:

1. It could be that your siRNA has modified ends and hence ligating the universal primer-oligo to the siRNA for cDNA synthesis priming is very inefficient. You'd have to check with the siRNA manufacturer about this.

2. your starting material is not enough; but 0.1 nM specific template sounds ok to me.

3. your qPCR is not optimal, e.g. too high or too low annealing temperature for your primers; or inefficient primers per se.

4. template in qPCR is not diluted and hence may inhibit qPCR - its usually recommended to dilute it at least 5-10x. We use 50x diluted cDNA and 5 uL of that are added to 15 uL qPCR reaction mix. This way we minimise pipetting errors

5. the qPCR cycling profile is not optimal for your primers. Check the Tm of your primers and the recommendations of your qPCR kit - some use Tm +/- x °C, depending on the buffer system. Also, check that your annealing time and elongation time are as recommended. Some qPCR kits recommend 2-step cycling. However, if the Tm is quite different from 60°C, you may need to use a three-step protocol.

6. have you tried to do qPCR with the endogenous/natural miRNA? Does that work well? Or is your synthetic miRNA designed for gene KD, then there will be no natural siRNA with the sequence. Still you could try amplifying another natural miRNA. If that works normal then you can at least exclude that it has do do something with your kit...

FINALLY: probably its best to repost your question as a new thread or post it in a discussion about qPCR of microRNAs.

Good luck and sorry that I could not give you more specific advice.

If you get consistently same values, I think its what it is. Can you try increasing the template concentrations and see if your trend remains consistent? I used to work with anti-microbial peptides and at 'no infection' conditions, I would get very high Ct values, similar to yours. As in ur case, NTC never showed me amplification, I used these values.

Christoph Metzendorf thank you for the suggestions. All my protocols are as per the kit I'm using. I've kept a range of annealing temperatures and the target concentration also seems good. But the synthetic oligo has it's 5' end modified. But in case of miRNA to cDNA conversion using stem loop primers, as the primer attaches from the 3'end and then proceeds for elonagtion I think the modified end may not be a problem. Also I did try to run the PCR with total RNA (1µg) as the starting material and got similar results while there are people working on the same miRNA using the same kit, getting values under 30. I have also posted the question as a separate thread. Also I tried making fresh stocks of all my primers, the nuclease free water that I'm using and also used a freshly isolated RNA (260/280= 2.1), but still nothing got better.

Bhagyashree Kaduskar thank you for your answer, I did try to increase the template concentration and have used up to 100nM of synthetic oligos and 1µg of total RNA but the values are still all above 40. Can you refer any paper which has used such high values please?

Arti Tyagi - concerning the 100 nM. It would be good to know whether CTs vs conc for 100 nM, 100 pM ... give you a good regression line (e.g. good fit, R^2 close to 0.99). Also, NTC (qPCR mix + water instead of sample) is only telling you if your primers do something funny by themselves or if you have contamination. -RT is the more relevant negative control, usually.

If others report CT under 30 (how much below? CT 29.9 would be below) for the same siRNA, same concentrations/total RNA amounts, same cycler, same kits, same program, same primers...... then you'd expect similar results. If you use different primers, probes etc it can change the efficiency of the qPCR, resulting in different CTs.

I guess qPCR with primers against mRNAs works fine? If so, you can exclude the qPCR program (at least in general) and master mix, if its the same for all your qPCRs.

I can only recommend to try normal trouble shooting steps, eg. dissect the different steps as much as possible to figure out which part goes wrong.

Christoph Metzendorf yes I understand that and thank you for your suggestions. The CTs under 30 are usually between 25-29 for the same miRNAs, kit, instrument and template concentrations. I've actually tried with a -RT control as well and both my NTC as well as -RT controls sometimes given Ct values as NA but sometimes theytoo give a Ct comparable to the target (in 40s or 50s) so the data is definitely not reliable. I'm also attaching images of my melt curve in two different trials (i usually get similar curves), can you please have a look at them too?

Hm, your melt curves are not complete - usually the temp should go up to your PCR-systems denaturation temperature; e.g. 95°C or 98°C to completely melt everything in your samples. Should look more or less like this (except for that "shoulder" problem https://www.researchgate.net/post/qPCR_melting_curve_analysis_shows_a_shoulder_on_BOTH_sides_of_the_curve_What_does_this_mean

In any case, melt curves will only tell you whether the products are similar/identical or not. It will not let you know if its the right thing. If you get the same product with your -RT then probably its the wrong product; if you get another product with -RT than with your samples, it is more likely that what you get is the real thing, but it could be something unspecific as well, just with different Tm ... .

I think your problem might be too complex to tackle via the web. It could be anything from wrong settings/programming of the thermocycler, to pipetting, calculation or handling mistakes during cDNA synthesis. If there is anyone at your institute that is good at qPCR maybe ask for help from that person. Could root out general qPCR problems.

@Christoph, an update: I tried it with a different set of primers that I borrowed from another lab and I got CT values of around 19-20 while my negative controls (-RT and NTC came around 34-37. I'm also attaching the peaks along (I've modified the melt temperature range) where red is the controls.

@Christoph, but it's for snU6. I mean the primers that I got and these results are for snU6.

Arti Tyagi the amplification (+RT vs -RT/NTC) of the primers you borrowed looks fine. But its not a synthetic siRNA like the target you are having the problems with. So if its something with the chemistry of your siRNA, this does not help much, except for showing that in principle your RT and qPCR is working. Why its not working with the the synthetic siRNA must be related to the siRNA (chemistry), but you said its not modified at the 3´ end to which the adaptor is ligated. Hence, the only thing left that could cause the trouble could be your siRNA-specific primer. You could try different melt temperatures because changing the primer isn't an option with such short amplicons. The other alternative would be to change the adaptor, but probably thats no option as the adaptor is part of a kit and the kit only comes with one adaptor, right?