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Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption
Abstract:
We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.
Insights
Researchers purified low-abundance hepatic messenger RNAs (mRNAs) using polysome immunoadsorption. This method efficiently isolates specific mRNAs for ornithine transcarbamoylase, propionyl-CoA carboxylase, and cystathionine beta-synthase.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Low-abundance hepatic mRNAs are challenging to isolate using conventional methods.
- Understanding gene expression requires efficient purification of specific mRNA transcripts.
Purpose of the Study:
- To develop and validate a method for purifying low-abundance hepatic mRNAs.
- To enable the isolation of mRNAs encoding key metabolic enzymes.
Main Methods:
- Polysome immunoadsorption using specific antibody-polysome complexes.
- Immobilization on protein A-Sepharose, followed by washing to remove non-specific polysomes.
- Elution of specific mRNA with EDTA and isolation via oligo(dT)-cellulose chromatography.
Main Results:
- Purified mRNAs for ornithine transcarbamoylase, propionyl-CoA carboxylase beta-subunit, and cystathionine beta-synthase precursors.
- Achieved purification folds ranging from 450- to 6,300-fold.
- Isolated mRNAs representing 0.20%, 0.02%, and 0.015% of total hepatic mRNA.
Conclusions:
- Polysome immunoadsorption is an effective technique for isolating low-abundance hepatic mRNAs.
- This method facilitates the study and cloning of cDNAs for low-abundance transcripts.
- The procedure holds promise for isolating other rare mRNA species.