Measurement of mRNA Decay in Mouse Embryonic Fibroblasts

Bio-protocol
Lian-Qun QiuPerry J Blackshear

Abstract

mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in the former case. This protocol is suitable for studying primary cultured cells and stable cell lines derived from transgenic mice and their respective controls, and provides for direct comparisons of mRNA decay rates in otherwise identical cells with and without the gene of interest.

References

Feb 4, 2009·Molecular and Cellular Biology·Thierry J HornerPerry J Blackshear

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Citations

Feb 24, 2019·The Journal of Immunology : Official Journal of the American Association of Immunologists·Emer P ReevesNoel G McElvaney
Jun 28, 2020·The Anatomical Record : Advances in Integrative Anatomy and Evolutionary Biology·Dan WangJinhua Li

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