Determination of enrichment factors for modified RNA in MeRIP experiments
| Autor: | Mark Helm, Franziska Weichmann, Gunter Meister, Jasmin Hertler, Aurellia Galliot, Regina Feederle, Kaouthar Slama |
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| Rok vydání: | 2019 |
| Předmět: |
Models
Molecular Adenosine Absolute quantification Methylation Protein Structure Secondary General Biochemistry Genetics and Molecular Biology Viral Proteins 03 medical and health sciences Adenosine Triphosphate RNA modification Escherichia coli Humans Immunoprecipitation Protein Interaction Domains and Motifs Nucleotide RNA Messenger Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences Messenger RNA Cell-Free System Chemistry Elution 030302 biochemistry & molecular biology RNA DNA-Directed RNA Polymerases Biochemistry Immunoglobulin G Isotope Labeling Chromatography Thin Layer Phosphorus Radioisotopes Protein Binding |
| Zdroj: | Methods. 156:102-109 |
| ISSN: | 1046-2023 |
| DOI: | 10.1016/j.ymeth.2018.10.020 |
| Popis: | In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m6A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m6A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry 32P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels. |
| Databáze: | OpenAIRE |
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