Efficient recovery of functionally intact mRNA from agarose gels via transfer to an ion-exchange membrane.
A simple method is described for the efficient recovery of intact mRNA from high resolution agarose gels. Fractionation of RNA is accomplished by gel electrophoresis under denaturing conditions using methylmercuric hydroxide. The RNA in the gel is then transferred electrophoretically to a diethylaminoethyl (DEAE)-membrane. After reversing the methylmercuric modification of the RNA, the membrane is sliced into narrow sections and the RNA is eluted at 65 degrees with a high ionic strength buffer containing 6M guanidine hydrochloride. RNA isolated by this procedure is suitable for subsequent enzymatic reactions, including in vitro translation and reverse transcription. The major advantages offered by this procedure are: 1) The membrane-bound RNA is a replica of the high resolution fractionation pattern achieved in the gel. 2) The immobilization and concentration of RNA and the removal of gel matrix contaminants are all accomplished in one step. 3) Small quantities of RNA are efficiently recovered and are suitable for subsequent biochemical manipulations. The method is of general utility for any biological system. We have applied its use to the fractionation, recovery, and analysis of mRNA from Xenopus liver and have identified cDNA clones complementary to albumin mRNA.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D004247:DNA / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005260:Female; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D014981:Xenopus
D012333:RNA, Messenger; D004247:DNA; D012194:RNA-Directed DNA Polymerase