Hello everybody!
We are trying to isolate DNA/RNA from thin primary FFPE melanomas. We use a protocol based on silica columns for the isolation of DNA/RNA from the same specimens.
Although the DNA that we get seems to work fine on subsequent enzymatic reactions (PCR, sequencing), we encounter inhibitory problems with the RNA samples. In particular, it seems that RNAs are contaminated with melanin. The amount of RNA that we have is very limited, 30 μl with a concentration of 20-50 ng/μl.
Has anybody used the CTAB-urea method or a column based method for removal of melanin from RNA preparations? Our problem is the very limited amount of the starting RNA.
Thanks a lot,
Olga
I wonder if RNA isolation (clean up) with Chelex 100 resin could allow you to work with a less melanin-contaminated preparation. I found the following protocol that illustrates RNA clean up with Chelex 100.
C S Edwin Shiaw, M S Shiran, Y K Cheah, G C Tan, A R Sabariah (2010). Evaluation of DNA and RNA extraction methods. Med J Malaysia 2010 Jun;65(2):133-7.
Dear Oscar, thanks a lot! I'll check the protocol that you suggested.
Olga
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