So have isolated RNA from FFPE. Originally our isolations were performed using the Qiagen AllPrep kits. Now we are using the RNA/DNA STORM kits from Cell Data Sciences.
We have a CORE that takes our RNA samples and generates library material using Illumina's kit: RS-122-2201/2, TruSeq® StrndTL RNALT RboZroHMN/Mse/Rat. We are using ribo-depletion and have asked for them to increase the PCR cycles from 8 to 12. We are also inputting between 500-750 ng of RNA.
Some times our samples work. Some times they don't (mostly they don't).
We have tried to correlate results between input ng, ratios (260:230 and 260:280), DV200 and RIN. We don't see any correlation over hundreds of samples indicating which QC is the better value to determine which will work.
We recently submitted 144 RNA samples of which 72 were processed but only 4 were successful. (NOTE: these were all prepared by the Qiagen AllPrep kits.)
We have the option of adding more cycles of amplification but we are concerned about duplication.
Any thoughts on how we can improve our library yields? I know that the CORE has been communicating with Illumina, but if there are groups out there, working with FFPE material and have tricks they are willing to share, I would greatly appreciate it.
Thanks.
We have a CORE that takes our RNA samples and generates library material using Illumina's kit: RS-122-2201/2, TruSeq® StrndTL RNALT RboZroHMN/Mse/Rat. We are using ribo-depletion and have asked for them to increase the PCR cycles from 8 to 12. We are also inputting between 500-750 ng of RNA.
Some times our samples work. Some times they don't (mostly they don't).
We have tried to correlate results between input ng, ratios (260:230 and 260:280), DV200 and RIN. We don't see any correlation over hundreds of samples indicating which QC is the better value to determine which will work.
We recently submitted 144 RNA samples of which 72 were processed but only 4 were successful. (NOTE: these were all prepared by the Qiagen AllPrep kits.)
We have the option of adding more cycles of amplification but we are concerned about duplication.
Any thoughts on how we can improve our library yields? I know that the CORE has been communicating with Illumina, but if there are groups out there, working with FFPE material and have tricks they are willing to share, I would greatly appreciate it.
Thanks.
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