Enrichment of poly(A)+ mRNA using immobilized oligo(dT).
This protocol describes a method for enrichment of poly(A)(+) messenger RNA (mRNA) using immobilized oligo(dT). It takes advantage of the fact that the vast majority of eukaryotic mRNAs contain a 3' tail of polyadenylic acid added post-transcriptionally. It relies on the ability of these tails to form stable hybrids with oligo(dT) under high-salt conditions. The hybrids are destabilized when salt is absent, allowing mRNAs to be recovered. This protocol is effective in removing the bulk of non-mRNA RNAs. A drawback of the technique is that it does not enrich for mRNAs that lack poly(A) tails (e.g., normal histone mRNAs or deadenylated mRNAs whose poly[A] tails have been removed during mRNA turnover). Several commercially available kits can be used for selection of poly(A)(+) RNAs. However, they are expensive and do not perform any better than "homemade" reagents using (dT) cellulose. One obvious advantage of this method is that mRNAs are enriched ~50-fold. Accordingly, detection by Northern blotting or reverse transcriptase polymerase chain reaction (RT-PCR) is considerably more sensitive.
Rio
Donald C
DC
Ares
Manuel
M
Hannon
Gregory J
GJ
Nilsen
Timothy W
TW
D011232:Chemical Precipitation; D008967:Molecular Biology / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*; D011061:Poly A / Q000378:metabolism*; D012333:RNA, Messenger / Q000302:isolation & purification*
D009841:Oligonucleotides; D012333:RNA, Messenger; D011061:Poly A