mRNA Stability Assay Using transcription inhibition by Actinomycin D in Mouse Pluripotent Stem Cells

Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional even...

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Published inBio-protocol Vol. 8; no. 21; p. e3072
Main Authors Ratnadiwakara, Madara, Änkö, Minna-Liisa
Format Journal Article
LanguageEnglish
Published United States Bio-Protocol 05.11.2018
Bio-protocol LLC
Subjects
Biology
Methods
Actinomycin D
mRNA half-life
Transcription inhibitors
mRNA decay
mRNA stability
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Summary:Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs in cells. The mRNA stability within cells can be measured indirectly by analyzing the mRNA half-life following transcription inhibition, where changes in mRNA levels are assumed to reflect mRNA degradation. Determination of mRNA half-life as a measure of mRNA stability is useful in understanding gene expression changes and underlying mechanisms regulating the level of transcripts at different physiological conditions or developmental stages. The protocol described here presents the analysis of mRNA decay as a measure for determining mRNA stability after transcriptional inhibition with Actinomycin D treatment in control and SRSF3 depleted mouse induced pluripotent stem cells (iPSC).
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Current address: Hudson Institute of Medical Research, Melbourne, Australia
ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.3072