Dear researchers,
I have ordered customized primers for ticks candidate miRNAs as these miRNAs are not available in any company. I have used a miRNA-specific MystiCq cDNA synthesis kit for cDNA synthesis and it has quite a good concentration. I have made qPCR with Maxima Sybr green with 10 fold dilution for efficiency curve which gave not a good result for 3 miRNAs (Ct 35-46, maximum undetermined), however with one miRNA average ct 26.66, 28.84, 35.31, 38.57, 41.50 and efficiency curve was 79.39. I have also adjusted annealing temperature using tm calculator from Thermo Scientific. Can anyone suggest what I could do to get a better efficiency curve? Should I also really need to change qPCR kit specific just for miRNA to get consistently acceptable ct and efficiency curve results for all miRNAs?
Thank you in advance for your answers.
Parwez
Hi, Parwez Ahmad
Please try temperature gradient during your PCR and do not forget about melt curves after the run. It could help.
In addition, try several mastermixes for PCR and do the same (temperature gradient and melting).
Use 2 step protocol during cycling
1 step +95oC (usually 10'' is enough)
2 step +55-65OC gradient (I use 59'')
Or redesign your primers....these are the easiest way.
Good luck.
What primers are you using during the reverse transcription step? Did they come with the RT kit or did you design them? Also, how do your melt curves look?
Hi Laura, I am using an oligo-dT adapter primer to convert poly-A tail miRNAs. They come with the MystiCq cDNA synthesis kit. For qPCR, I am using another kit that is available in our lab called Maxima Sybr Green together with a designed primer designed by a software called miRprimer2 and manufactured by a company. The melt curves are not consistent and look quite bad as per my understanding. Please suggest as per your experience if you have some idea to optimize it/ if it's really important to have both cDNA synthesis kit and qPCR kit specific for miRNA and from the same company. Thank you.
Hi Parwez, I can't think of any reason why it would be important to use reagents from the same manufacturer. It's definitely possible to do kit-free miRNA qPCR. I think if you can diagnose and fix the issue with your primers that your assay will work fine.
I'm not familiar with the software you're using but I had a look on github and it sounds like it is designing both a forward and reverse primer for qPCR for your miRNAs? What does the reverse primer look like?
Thank you Laura. Yes, I think so too as we have already CDNA prepared by miRNA specific kit and I don't see any logic to keep working with miRNA specific qPCR. Yes, I have forward and reverse primer which I am working with. I see reverse primer with a repetitive 15 T sequence in middle too.
Would you be able to post the primer sequences? It's much easier to troubleshoot qPCR when you can see what you're working with :)
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