I decided to go out on a limb and do RNA seq in a tissue that, to my knowledge and extensive research, has never been done before. I knew it was a risk based on the tissue type… well sent in 20 samples. 18 have an RIN between 1.5 and 2.5. And 2 have acceptable RINs. Well I know the 1.5-2.5 samples are basically useless so now I’m left thinking… do I go ahead and sequence the 2 that did pass QC? What would be the point? I can’t compare it to anything. And I’m not looking to do a full manuscript from this, I was hoping more for an abstract…. Could I use the 2 okay samples to say hey this is never been done before but has some potential! Look what genes have the highest counts! I dunno. Feeling defeated and looking for hope but know there probably isn’t much.
Illumina has some options for low quality samples like formalin-fixed parrafin embedded tissues here which could fit your use case. Ribosomal RNA depletion would be recommended over poly-A enrichment. Targeted sequencing has worked for us with partially degraded samples. You can include RNA quality as a covariate in the statistical analysis.
What is it then good for beyond your personal curiosity? An abstract won't give you any new fundings and by itself is not publishable to any proper journal. It is also not impressive on your CV.
It seems to me you are looking for a "go ahead" to continue with these samples but as you say it is not very meaningful to me. RIN of 1.5 is really low, it's essentially a degraded transcriptome. Even if you manage to sequence that, it's more rubbish than anything else.
Why not spending time optimizing a tissue extraction protocol to get viable cells, maybe together with a FACS panel of important celltypes and then a bulk RNA-seq of some sorted populations. This would be publishable. Not high most likely, but it could be a solid methods paper -- given that it's true that this tissue is known to be difficult and no proper methods for what you're doing with it have been established. But a poor RNA-seq sample along won't give you much I guess. Always think ahead, like, what is the analysis? It essentially comes down to reporting a list of genes without context because that is what RNA-seq without comparison or phenotype gives you.
Thank you for your reply!
My program is super intense when it comes to writing abstracts and then going to conferences. So that is why I mentioned the abstract.
And I totally agree that focusing more time on optimizing a protocol for this tissue is what needs to be done. But overall was wondering if there was anything I could do with what I have. Another factor is the funding used for these needs to be spent relatively quickly so that’s another reason why I figured why not go ahead with the couple of samples that do have an acceptable RIN.