I need to know some explanation of methods that use to cDNA synthesis from miRNA?
Hi Payman,
There are 2 main methods to generate cDNA from miRNAs:
1- PolyAdenylation method: In this method, you first use poly A polymerase enzyme to add lots of A bases at the end of miRNAs. Then you use a universal primer so that you can have a pool of total cDNA. This cDNA can be used directly for evaluating all miRNAs by qPCR (SYBR Green-based qPCR).
2- Stem-loop method: In this method, you add a commercially available stem loop to generate cDNA from specific miRNA. As compared to previous technique, here you do not have a total cDNA. Then you can use this particular cDNA for TaqMan-based qPCR.
The advantage of the first method is that you have total cDNA, so you can screen different miRNAs with a common cDNA. However, since the qPCR method is SYBR Green-based, the accuracy is not always acceptable. This is exactly the advantage of the second method, in which you can evaluate the expression of miRNA accurately and confidently.
Please have a look at the attached files.
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