Preparation of poly(A)+ RNA.
Most messenger RNAs contain a poly(A) tail, while structural RNAs do not. Poly(A) selection therefore enriches for messenger RNA. The technique has proved essential for construction of cDNA libraries. It is also useful when analyzing the structure of low-abundance mRNAs. Removing the ribosomal and tRNAs from a preparation increases the amount of RNA that can be clearly analyzed by S1 analysis, for example, thus allowing detection of a low level message. This protocol separates poly(A)+ RNA from the remainder of total RNA, which is largely rRNA and tRNA. Total RNA is denatured to expose the poly(A) (polyadenylated) tails. Poly(A)-containing RNA is then bound to oligo(dT) cellulose, with the remainder of the RNA washing through. The poly(A)+ RNA is eluted by removing salt from the solution, thus destabilizing the dT:rA hybrid. The column can then be repeated to remove contaminating poly(A)- RNA.
Kingston
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Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012996:Solutions
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012996:Solutions