Enrichment of poly(A)+ mRNA using immobilized oligo(dT)
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Updated: Jun 10, 2026
An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes
Published on: August 18, 2018
Introduction:
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.
Insights
This protocol enriches poly(A)-tailed messenger RNA (mRNA) using immobilized oligo(dT), significantly improving detection sensitivity. The method offers a cost-effective alternative to commercial kits for isolating eukaryotic mRNA.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Most eukaryotic messenger RNAs (mRNAs) possess a poly(A) tail.
- This polyadenylation is crucial for mRNA stability and translation.
- Certain mRNAs, like histone mRNAs, naturally lack poly(A) tails.
Purpose of the Study:
- To describe a method for enriching poly(A)-containing mRNA.
- To provide a cost-effective alternative to commercial mRNA enrichment kits.
- To enhance the sensitivity of mRNA detection methods like Northern blotting and RT-PCR.
Main Methods:
- Utilizes immobilized oligo(dT) to bind the poly(A) tails of mRNA.
- Relies on high-salt conditions to stabilize mRNA-oligo(dT) hybrids.
- Releases bound mRNA by removing salt, separating it from non-polyadenylated RNAs.
Main Results:
- Achieves approximately 50-fold enrichment of poly(A)(+) mRNA.
- Effectively removes the majority of non-mRNA RNA contaminants.
- Demonstrates increased sensitivity for downstream applications such as Northern blotting and RT-PCR.
Conclusions:
- Immobilized oligo(dT) provides an efficient method for poly(A)(+) mRNA enrichment.
- This protocol is a cost-effective and sensitive approach compared to commercial kits.
- The enrichment facilitates more sensitive detection of specific mRNA transcripts.
