0000000000022598
AUTHOR
Franziska Weichmann
showing 2 related works from this author
Determination of enrichment factors for modified RNA in MeRIP experiments
2019
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…
Validation strategies for antibodies targeting modified ribonucleotides
2020
Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2′-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abun…