6533b7cefe1ef96bd12570b3

RESEARCH PRODUCT

Determination of enrichment factors for modified RNA in MeRIP experiments

Mark HelmFranziska WeichmannGunter MeisterJasmin HertlerAurellia GalliotRegina FeederleKaouthar Slama

subject

Models MolecularAdenosineAbsolute quantificationMethylationProtein Structure SecondaryGeneral Biochemistry Genetics and Molecular BiologyViral Proteins03 medical and health sciencesAdenosine TriphosphateRNA modificationEscherichia coliHumansImmunoprecipitationProtein Interaction Domains and MotifsNucleotideRNA MessengerMolecular Biology030304 developmental biologychemistry.chemical_classification0303 health sciencesMessenger RNACell-Free SystemChemistryElution030302 biochemistry & molecular biologyRNADNA-Directed RNA PolymerasesBiochemistryImmunoglobulin GIsotope LabelingChromatography Thin LayerPhosphorus RadioisotopesProtein Binding

description

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.

yearjournalcountryeditionlanguage
2019-03-01Methods
https://doi.org/10.1016/j.ymeth.2018.10.020