TRIzol purification precipitate out everything BUT the RNA Two common methods for separation of RNA from DNA Selective precipitation of RNA using Lithium Chloride LiCl Extraction with phenol buffered at an ID: 1043136
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1. RNA purification1LiCl purification: precipitate out the RNATRIzol purification: precipitate out everything BUT the RNA
2. Two common methods for separation of RNA from DNASelective precipitation of RNA using Lithium Chloride (LiCl)Extraction with phenol buffered at an acidic pH (~4.5)NOTE: DNases are typically NOT used to remove large amounts of DNA from RNA because DNase is too expensive and is not efficient enough for that purposeSome protocols that have a small amount of DNA contamination might specify using DNase 2
3. Principle of Selective precipitation of RNA using Lithium Chloride (LiCl)Li+ ions form a Li+– RNA- complex with no net charge, which precipitates out of the solutionSingle stranded nature of RNA and the 2’ OH group facilitate thisThe procedure is done using high concentration of LiCl and incubation on ice to promote the precipitation of the RNA saltBest for RNAs above 100 bases long 3
4. Selective precipitation of RNA using Lithium Chloride (LiCl)Add 7.5M LiCl to your crude lysate (having spun out the cell debris)Incubate at -20oC (i.e. in the freezer) for about 30 min (or longer)Centrifuge to collect the RNA pelletDesalt with very cold (-20oC) 70% ethanol (look at the LiCl concentration and consider the importance of this step)Resuspend in nuclease free water or other solution4
5. Extraction with phenol solution buffered at acidic pHRNA is most stable at pH 4-5, DNA is most stable pH 8Combine extraction buffer and organic solvent:Suspend cells, homogenized or powdered tissue in acidic phenol solution – (phenol + water with buffer at acidic pH)Add chloroform, mix well, and centrifuge Separates into lower organic phase and upper aqueousCollect the upper - aqueous - layer that contains RNAMay be a noticeable interphase between the two layers - avoid touching this with the pipette!5
6. Crude lysate containing nucleic acids and other cell constituents- proteins denatureadd chloroformand shakeCentrifugeThe aqueous phase contains water-soluble molecules, including RNAHigh-molecular weight DNA fragments and proteins in the interphase.Collect aqueous phaseTRIzol variant of acidic buffered phenol, guanidinium thiocyanate, chloroform extractionOrganicAqueousDNA, protein, cell wallMix tissue powder with trizol and vortexisopropanolsalt precipitationcentrifugationRNA pelletwash pelletwith 70-85% ethanoldry pelletresuspend in nuclease freewaterRNA solutionTrizol (trireagent)monophasic solution of1. guanidinium salt2. phenol3. buffer with low pH6
7. Once you have an RNA solution, there is a risk of: Some RNase remaining (from the tissue originally) RNase introduced (breath, reagents, equipment etc.) The solution: Common to add protein inhibitors of RNase (RNAsin is one of them) Commercially available, but expensive Sensitive to denaturants (heat, chaotropic salts, phenol/chloroform Add only at end of procedure7