RNASeq from total RNA with a RIN under 8

[jo_mason]

Hi,
Has anyone got any experience in carrying out RNASeq from starting material with a RIN less than 8?
How well did it work?
We have some samples that continually fail to to give a RIN above 8, we are pretty sure it's because of the starting material we are extracting from, as others are fine. I'm pretty tempted to just give them a go and see how it turns out, but does anyone else have any experience of this?
How low can the RIN get before the sequencing is badly affected?
If anyone is willing to share their experiences it would be very much appreciated!
Thanks,
Jo

[pmiguel]

The effect of degradation of RNA has on sequence depends on the ribosomal RNA depletion method and the first strand synthesis method.

If either of these steps relies on the polyA tail of mRNA, then degradation is a problem--your sequence will be biased towards the 3' ends of transcripts.

TruSeq RNA prep kit? Could be a problem: uses a polyA binding to deplete ribosomal. We had success using Ribo-Zero (Epicentre) to pull ribosomal RNA from the prep prior to merging back into the TruSeq protocol at the "Elute Frag Prime" step. Note that your RNA may already be partially fragmented, so you may want to back off on your fragmentation times. We used 4 minutes instead of 8 and that was plenty.

--
Phillip

[peromhc]

I have dont RNAseq with RIN in the high 6's, poly-a prep using Illumina reagents.. Some rRNA contam, but not terrible.

Why don't you tell us about your RNA, show a bioanalyzer pic, etc.