Hi,
I am working on circulating RNAs available in human plasma. After cDNA synthesis using TaKaRa Kit I performed qPCR (with amplicon) with GAPDH and U6 primers, but the result is a bit confusing and seems it needs a set up which makes me be in trouble as I don not know how can I solve this problem. The melt curve picks differs case by case and we can not access a fixed pick. Is there any one here with the same experience like this ( there are attaches)? How can I remove or decrease the pcr inhibitors?
Hello Nahid
I would like help you. Your study of circulating RNA in human plasma are about microRNAs or genes?. In plasma microRNAs are more stable with mRNA.
Regards
As Oscar as pointed out, typically RNA gets degraded (RNase) in blood as RNA is a very fragile molecule outside of the cell. However there are exceptions for this (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515835/). Typically blood/serum profiling involves DNA and microRNA, is that why you've used U6? How did you make your cDNA (which kit) for U6 and were the primers custom design or validated by the company?
Yes there is definately non-specific amplifaction as suggested by the melt curves.
For the second picture, perhaps you are getting gDNA amplification. See figure 9.9 of link below
http://www.sigmaaldrich.com/technical-documents/articles/biology/assay-optimization-and-validation.html
are you working on miR in plasma as such or on detection of miR in cell free plasma?
If the latter is the situation, so you have first of all to spin the serum or plasma at 14000-16000xg and work quickly or store at -80 or in liquid Nitogen, we found non specific findings like yours if do not handle the sample in ideal conditions like when you work on mRNA.
the inhibitors can best you get rid off them by diluting your cDNA 1:50-1:100 and use the diluted samples in the qPCR applications. Also try to change the annealing temperature and observe the effect of these on melting curve analysis. If you noticed no improvement, i suggest to try to re-design an new primer set.
Thanks for all
I synthesized cDNAs with TaKaRa kits and I am going to profile mRNAs and lncRNAs and these attaches are my pilot works. U6 primers are validated by company. U6 is very popular with scientists in profiling miRNAs. Why isn't it used for mRNA profiling?
I couldn't understand how to eliminate the inhibitors Dr. Abdulrazzaq Hasan. Would you please clarify it?
Best
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