hi Fatma Abdel Wahab

General steps: RNA/miRNA extraction-> cDNA synthesis-> qRT-PCR (SYbr/Taqman) using miRNA primers.

->RNA from plasma and serum is normally fragmented RNA or small RNA, mainly miRNA, and is either bound to proteins or contained inside extracellular vesicles. You can look Norgen exosomal RNA purification mini kit. Even Qiagen and Thermo have miRNA extraction kits, whichever you find cost-effective, you can go for it.

If total RNA is from cell lines or tissues, then you can go with Qiagen miScript RT kit II. The kit basically converts all miRNA present in particular RNA samples into miRNA-specific cDNA. That cDNA can be used directly with SYBr based qRT-PCR. So, here if you get different miRNA primers of your interest, you can analyze them with that miRNA-specific cDNA with SYBr based qRT-PCR.

We have used this method in our study. Article Inhibition of breast cancer stem-like cells by a triterpenoi...

NOTE: I. Here using that Qiagen cDNA kit (Qiagen miScript RT kit II) only, you can go for SYBr. Since many including me found that the Thermo miRNA advanced cDNA kit generally does not work for SYBr, so you have to get the Taqman probe.

Hope this may help you.

Regards

Saurabh

Actually, it’s not effective process to extracat miRNA, cause most extraction kits of total RNA designed for almost long RNA not for short length like miRNA. So u have to use a specific miRNA kits.

Hi Fatma Abdel Wahab

Most RNA extraction kits do not recover RNA molecules smaller than 200 nts in length, such as miRNA. So I would not recommend such a kit for miRNA extraction. There are several kits that are now available for the purification of total RNA along with miRNA/small RNA (like the miRNeasy series from Qiagen or the miRVana series from Thermo Fisher). You should look into these.