The Cts look too early for microRNA don't you think? Amplification of 18S would/could have a Ct such as that. I've done some microRNA via SYBR green method (with universal reverse primers), I don't think I saw anything earlier than 20 Cts and that was for the microRNAs that had relatively high read count with NGS.

Fatemeh Ranjbarnejad

Are you sure there is no problem with the primers you use with those samples?

You can put it on gel electrophoresis and see what the result is.

You don't have a positive PCR reaction. It means you don't have a target in your PCR mix or your PCR reaction isn't well optimized.

First check up your primers, it seems that there is a hairpin or dimer. re design them and run RT PCR again with + PCR reaction

Look at the scale of the Y-axis on your graphs. You are getting no amplification. This is just background noise.

Add in a good positive control and try again.

Good luck!

Thank you everyone for your valuable advice. actually I had this problem in every three miRNA that in some samples CT value was low around 12 and in other samples and duplicates I did not have amplification and CT value. Despite the low CT value, could this be due to primer dimer? In this case, do I need to redesign the primer or can it be solved by optimizing the temperature?

Katie A S Burnette

dear Dr. Burnette

can be noise the samples that have ct value near to 12 for example?

Can Kiessling

Dear Dr. Kiessling .Thank you for your reply.

actually the CT values are very low and I doubt their accuracy. I have seen even lower CT like 6 and an important point is that I got ct value 20 in NTC! .

The fact that your NTC got relatively early amplification is worrisome. Since you are using SYBR green, something (other amplicons from other people/assays) aerosolized could be giving non-specific amplification positivity which would make sense. An early Ct positive result for NTC is a no-go, a clear red line in the sand. It has deep implications, your lab may need to take action if there is aerosolized contamination. I've seen it happen in my previous jobs.

The fact that you don't have consistent technical replicates means the issue is not "sample-dependent" and must be something shared by the reactions. What does your standard curve look like? Your positive controls?

Katie A S Burnette Thanks a lot dear Dr. Actually, I don't have a standard curve yet. Is this section mandatory?

Can Kiessling

Thanks a lot dear Dr. I will check my reagents for contamination, but other researchers in our lab have no problem with NTC

Other researcher don't have to have the same problem, if you have it then it's sufficient enough to stop & warrant inspection. Do you think it would be logical to ignore the fact that NTC was amplification positive even though other people in the lab don't have it, what would that mean?

If you want to publish anything, then you have to have a standard curve. That’s how you calculate the primer efficiency, which must be 95-105%.

Katie A S Burnette Yes then it must be done and I will do it. thank you very much.

Can Kiessling You are right, I will check all factors to fix this problem. Of course, I meant that because other people who work in our laboratory do not have problems with NTC, then the possibility that my materials are contaminated is more than that there are contaminated aerosols in the laboratory environment, and maybe it is better that I first check my own reagents. Thank you very much for your reply

Your observation about other people not having NTC amplification problems could be helpful in narrowing the source. These other people, are they preparing the PCR reactions somewhere else from you? Are you all preparing PCR reactions in the same room? Depending on the answers, you can quickly narrow down the physical location of your troubles.

Can Kiessling Yes, we work in a common hood, which, of course, is sterilized with disinfectant solutions and UV every day before and after work.

Based on those answers, where do you think the NTC signals are coming from? Could there be a reagent that is contaminated, a buffer, dNTPs, Taq mix etc? These are not small amplifications, anything around 20 Ct is relatively high expressed. Can you do a NTC reaction like this, couple of replicates that you prepare with your own reagents, then ask a colleague in the lab who doesn't have this problem to do couple replicates with their own reagents. You should run your reaction with your colleagues reactions on the same PCR run to detect signal. You can use separate strip tubes (0.2 mL, 0.1 mL or whatever is available), whatever suits you but just make sure to analyze the samples in the same run.

By the way, if you colleagues aren't using the same primers as you are (that would match an assumed aerosol contaminant), this doesn't necessarily still rule out aerosolized contaminants. We don't know the identity of this amplicon, if someone doesn't have primers to amplify this aerosolized contaminant then it might not be a problem for them. This is a hypothetical scenario. Also follow up the above experiment with this, give your reagents to a colleague who doesn't have the problem and let them do the same thing you are doing. If they're getting the same problem then you know something in relation to your reagents is triggering this. You can also give your primer for them to try, they can use their own mix and you give them your primer. If you give them your primer and they use their own mix, NTC amplification will give you why they don't see it with their own reactions but you see it assuming you use different primers to them.

Can Kiessling Dear Dr Kiessling

I will apply all your instructions and find the cause of this problem. Thank you very much for your valuable advice