Hi guys, I am trying to quantify the miRNA by using real-time PCR. After the RNA extraction, I need to add the poly A tails to mature miRNAs and I would assume that a poly A tailing kit should be able to do this. But after the poly A tailing, do I need to inactive poly A polymerase? Will the poly A polymerase affects downstream applications like reverse transcription and real-time PCR. I have this question is because the real-time PCR amplification curves after poly A tailing and reverse transcription look really wired to me and the CT values keep really low and when I diluted the sample, the changes of the CT values are not exponential if we assume that two-fold change in the concentration of c DNA equals 1 CT value change. Does anyone have this kind of experience? Thank you!
You can check this website.
http://www.thermofisher.com/nl/en/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/microrna-noncoding-rna-with-real-time-pcr/mirna-quantitation.html
I don't have any shares in this company ;-) but I like their assays. I use RNA isolated with TRIzol from patient derived leukemic blasts. Per microRNA you make cDNA specific for the microRNA you would like to quantify. Per cDNA reaction 5 ng of RNA was used. You can of course adjust this. This microRNA specific cDNA you use in the RT-qPCR. The assay of Life technologies consists of primers voor making cDNA and an primer/probe mix for the RT-qPCR. The RT-qPCR reaction is done in an 384-wells plate. Pleas read the instructions which reagents you need to order for this assay to work.
Hi Xingchen
You should purify your miRNA after PAP treatment because its buffer contains inhibitors that affect Reverse Trasncriptase.
Thank you so much for all of you guys' reply and i am doing the phenol-chloroform extraction and ethanol precipitation after the tailing then I will proceed to do reverse transcription and real-time pcr. I will keep you guys posted and let you know if i am able to solve the problem, thank you agian!
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