RNA-Seq and Poly(A)

[Bio.X2Y]

Hi folks,

I wonder if someone can help with a few beginner questions around poly(A) and RNA-seq?

We used the standard Illumina kit to purify poly(A) RNA for rna-seq. I'm trying to understand the repercussions of this for the various types of RNA.

mRNA
Most mRNA will get through to the reading stage. Is this correct?

rRNA
I've been told that some will get through. Is this because some rRNA is poly(A), or because some non-poly(A) RNA gets through.

ncRNA
Do non-coding transcripts tend to have poly(A) tails? I've getting mixed messages about this - perhaps it is an area that is not fully understood?

tRNA
Any ideas?

Thanks for your time!

[krobison]

WRT ncRNAs, this paper which found many large ones used polyA+ RNA, suggesting they are polyadenylated

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754849/?tool=pubmed

Nature. 2009 Mar 12;458(7235):223-7. Epub 2009 Feb 1.
Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.
Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, Cabili MN, Jaenisch R, Mikkelsen TS, Jacks T, Hacohen N, Bernstein BE, Kellis M, Regev A, Rinn JL, Lander ES.

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Comment in:

Nat Biotechnol. 2009 Apr;27(4):346-7.
Abstract
There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified approximately 1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFkappaB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.

PMID: 19182780 [PubMed - indexed for MEDLINE]PMCID: PMC2754849