For a microRNA expression study, if the volume of the starting source material is variable then is it justified to quantify and take equal amount of miRNA as templates for single strand cDNA synthesis (reverse transcription)?
Hi Amit,
Yes of course. To compare you must have an equal amount of microRNA, working with microRNA is similar to working on total RNA, and you can also use the total RNA for a microRNA expression study.
Amine
Hi. as microRNAs constitute a small fraction of total RNA it is almost imposible to quantufy their exact amount befor cDNA synthesis. thus as Amine said, researchers use equal amoumt of total RNA for each cDNA synthesis reaction. moreover if you are going to evaluate miRs expression alterations by relative quantification method, choosing a relaible internal control would be necessory for normalization.
As it was said before, use equal amounts of total RNA and make qPCR for your gene together with reference short RNA - people often use RNU6B. In our hands SNORD95 worked fine,
Best luck,
Bartosz Wojtas
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