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Old 04-11-2013, 12:41 PM   #1
acme
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Location: Minneapolis

Join Date: Apr 2011
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Default Preparing yeast RNA for Illumina HiSeq

Hi,
I've been trying to figure out how folks prefer to prepare total RNA from yeast for Illumina HiSeq. I prefer the rapid death and lysis phenol/phenol:ChCl3 provides over mechanical or enzymatic lysis and I will do a DNAse step and a column clean up. The RNAeasy protocol starts either with RNA resuspended in water for removing trace phenol or using the non-phenol methods. I've seen protocols and discussions on all the above steps, but no one that combines them all. Is it overkill to do one hot phenol extraction followed by phenol:chloroform:isoamyl alchohol then DNAse digest and clean up on a column? Is there any reason to think one shouldn't apply the aqueous phase from the P:C:I step directly to an RNeasy column (after removing the aq. layer and then adding ethanol as per the mfr direction)? The volume of the aq. layer + ethanol would be quite high and require multiple passes over the column. Would that lower yield too much?

I appreciate your thoughts.
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Old 07-19-2013, 08:53 AM   #2
acme
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So, I did the protocol in this article and it worked awesome. I recovered ~3-4 mg per sample (as protocol indicated I might).
http://www.ncbi.nlm.nih.gov/pubmed/20134426

No trouble whatsoever getting good RNA from the extraction part, but I did have a deal with the on-column DNAse digestion. Oddly, it wasn't necessarily degradation. The RNA I recovered from the column was smeared up the gel, not down it (non-denaturing, straight TAE electrophoresis) and I could not get reliable nanodrop readings.

Two thoughts. One is carryover of something from the column (e.g., chaotropic salt). In trying not to expose the column to any more than necessary, I didn't invert the column to wash the column itself. As a result, salt hanging out at the top may have carried through. The other possibility is that I overloaded the column. I just threw a whomping amount of RNA on it, not thinking about binding capacity. So I don't know which is too blame, but when I repeated the column step and targeted only 90 ug for loading and did invert the column to wash, the problem went away. I had awesome RNA that looked good on the gel, quantified on the nanodrop and passed Illumina QC well enough for my sequencing center.

42 samples. Looking forward to that pile of data.
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