I am preparing cell lysates in a lysis buffer containing Triton X-100 (a non-ionic detergent) and SDS (an ionic detergent). I want to extract RNA with phenol/chloroform/isoamyl alcohol. Will Triton X-100 and SDS partition into the aqueous phase with the RNA, or into the organic phase?
(The aim of my experiment is to isolate RNA without disrupting native secondary structures. I am avoiding chaotropic salts, column steps and precipitation steps. I will desalt and concentrate my RNA by buffer exchange on an Amicon 10K column and I'm trying to work out how much, if any, detergent I might have to wash out.)
While the detergents help to digest the protein and non nucleic acid components of the cell, adding phenol/chloroform further bring the RNA in the aqueous phase and the cell debris/lipids in the organic phase. You can centrifuge the aqueous phase/ employ ethanol extraction to purify nucleic acid from the aqueous phase.
phenol/chloroform/isoamyl alcohol extraction could use for buffer exchange, should be almost no detergent left.
The thing I think about is the definition of rna 2nd structure. I think the RNA will have a different (slightly) 2nd structure in different pH, buffer.
Hi Laura Leighton . Detergents, along with lipids, should be in the organic phase.
But, phenol/chloroform extraction is by definition a chaotropic procedure, so I'm not sure how you can determine what is the "native secondary structure" of your RNA after this step.
Thanks everyone for your input! :) I am fairly sure that the detergents effectively partitioned out of my aqueous phase during 2x extractions with PCI and 2x extractions with chloroform. The final aqueous phase had no tendency to foam up when shaken like solutions containing even low percentages of detergents do.
Steingrimur Stefansson I agree! The complete protocol involves cellular lysis and proteinase K digestion in a buffer containing Triton X-100 and SDS (chaotropic), then 2x PCI extractions and 2x chloroform extractions (chaotropic), followed by buffer exchange into HEPES with physiologic ion concentrations and equilibration at 37C. I would guess that the structure of RNA is mainly determined by the refolding buffer and equilibration step, and that I'll see very little difference due to the extraction method. But published papers highly recommend this 'gentle' RNA extraction protocol, so I think I will directly compare them and see!
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