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mutant is gone in cDNA


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#1 tashua

tashua

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Posted 08 January 2010 - 07:35 PM

Hi,
I found a missense mutation in DNA. Then I checked this mutation in cDNA using RT-PCR. mRNA was isolated from the Epstein–Barr virus immortalized lymphoblastoid cell line using RNeasy kit, mRNA was treated with Turbo DNase and was reverse transcribed into cDNA. Primers were designed on exon-exon junction. During the RT-PCR I also included minus-reverse transcriptase control. I saw specific band at all cDNA samples but not in minus-reverse transcriptase control. That means no DNA contamination. Then I sent it for sequencing, my mutation was in cDNA.

Later, I was able to get mRNA that was isolated directly from blood. I did exactly the same thing that I did above. When I got the seqs results, my mutation was gone.

Do you have any idea what happened? Why mutation was in mRNA from the cell lines and not in blood derived mRNA.
many thanks




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