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Old 01-31-2011, 10:18 AM   #1
daughart
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Location: Philadelphia PA

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Default Comparing Poly(A)+ selection methods - rRNA contamination, yield, etc.

My previous RNA-seq experience is with total RNA-seq, using RiboMinus to deplete rRNAs. Now we are considering mRNA-seq. We plan on using the Illumina dUTP paired-end library construction method with human RNA from whole blood (Leuko-lock). Our starting amount is 12-18 ug of total RNA, which is on the low end.

What are your experiences with Poly(A)+ selection and RNA-seq?

MicroPoly(A)Purist (considering our small starting amount) - do we need to do RiboMinus first, or two rounds of MicroPoly(A)Purist? What efficiency should we expect, both in amt. of recovered Poly(A)+ RNA and number of rRNA reads in the final sequencing?

What about DynaBeads oligo(dT) purification? Are there any other favorite methods? I would really appreciate any advice people can give, or if you can link to other posts. I've already gone through many of them, and there's a lot of mixed advice out there.
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Old 01-31-2011, 10:58 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Default

We get 2-3x higher yields with the old style, non-magnetic, oligo dT cellulose (batch hybe + spin columns) than using magnetic beads. No idea why. (PolyA Purist is the kit we currently use.)

That said there are enormous tissue/organism specific variation in polyA content of total RNA. So your best bet is to switch your research to working on fish gonads. polyA yields are crazy high!

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Phillip
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