miRNA sequencing from FFPE tissue - (May/24/2013 )
I'm working on sequencing miRNA from FFPE tissue. We've just run a trial using the Roche high pure total RNA extraction method and NEB small library prep for the Illumina HiSeq. Our results are a bit disappointing. We got a low percentage of reads aligning to the miRBase (around 1-3%!). Though this amounts to about 500,000 reads each sample there seems to be a lot of genomic DNA contamination (and mRNA, rRNA). The extraction method has a DNAase treatment step but clearly has not removed al the DNA. Does anyone have any experience with this? I was thinking about changing extraction kit to something like Qiagen miRNAeasy FFPE kit and adding a ribosomal RNA depletion step but am concerned that the NEB library adaptors are not specific to miRNAs as they have clearly ligated to lots of non-miRNA. Does anyone have any experience of this? What sort of read alignments were you getting? which library prep did you use? How much enrichment for miRNA did you do? Sorry for all the questions but am very disheartened and any help would be much appreciated.
No personal experience with small RNA deep sequencing. There are several papers on this, not sure if you have looked at them. Talking directly to the authors will help.
Sorry for the delay in replying have been away for a few days. Thnaks so much for the papers and advice. Is it reasonable to just email the corresponding author with these sort of questions?
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