Dear/ Madam
I am a little confused about extraction of microRNA from FFE block. whether total RNA is first extracted from FFE tissue and then miRNA is extracted from total RNA or Can I synthesize single strand cDNA for miRNA can be synthesised from total RNA.
hi,
I don't have much idea about FFE block.
But We have used the Qiagen miScript II RT Kit, where we used total RNA from cells (isolated via Trizol method), Kit converts all the miRNA present in the total RNA into miRNA-specific cDNA. That cDNA can be used for qPCR directly using SYBr green. So, you can look a similar kind of product from Qiagen for the FFPE block.
Regards
Saurabh
Hi Sevan,
You could use miRNeasy FFPE Kit from Qiagen to isolate RNA. It is especially designed to overcome formalin cross-linking and allows you to isolate both miRNA and total RNA. After isolating RNA, we use miRCURY LNA miRNA PCR kit from Qiagen to synthesize cDNA and perform RT PCR.
Hope this helps!
Thanks a lot Saurabh, Asemoloye and Shyambabu for your advice. I know miRNeasy FFPE kit and miRCURY LNA miRNA PCR kit are the best. Actually I can not get them, because there are not any distributors of Qiagen in my city. I have got hardly Norgen kit via a company far from my city.
best regards
sevan
If you use total you will get mostly ribosomal. You should size select first.
I agree, I kit-based isolation assay should work as expected (e.g. see miRNeasy FFPE Kit and others).
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