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.
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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