Evaluating the Stability of mRNAs and Noncoding RNAs.
Changes in RNA stability have an important impact in the gene expression regulation. Different methods based on the transcription blockage with RNA polymerase inhibitors or metabolic labeling of newly synthesized RNAs have been developed to evaluate RNA decay rates in cultured cell. Combined with techniques to measure transcript abundance genome-wide, these methods have been used to reveal novel features of the eukaryotic transcriptome. The stability of protein-coding mRNAs is in general closely associated to the physiological function of their encoded proteins, with short-lived mRNAs being significantly enriched among regulatory genes whereas genes associated with housekeeping functions are predominantly stable. Likewise, the stability of noncoding RNAs (ncRNAs) seems to reflect their functional role in the cell. Thus, investigating RNA stability can provide insights regarding the function of yet uncharacterized regulatory ncRNAs. In this chapter, we discuss the methodologies currently used to estimate RNA decay and outline an experimental protocol for genome-wide estimation of RNA stability of protein-coding and lncRNAs. This protocol details the transcriptional blockage of cultured cells with actinomycin D, followed by RNA isolation at different time points, the determination of transcript abundance by qPCR/DNA oligoarray hybridization, and the calculation of individual transcript half-lives.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 748-sala 1208, Sao Paulo, SP, 05508-900, Brazil.
Ana Carolina
AC
Ayupe
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 748-sala 1208, Sao Paulo, SP, 05508-900, Brazil. emreis@iq.usp.br.
Eduardo M
EM
Reis
D018929:Cell Culture Techniques; D003609:Dactinomycin / Q000494:pharmacology; D020869:Gene Expression Profiling; D020043:Genes, Essential; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry* / Q000302:isolation & purification*; D022661:RNA, Untranslated / Q000737:chemistry* / Q000302:isolation & purification*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D022661:RNA, Untranslated; D003609:Dactinomycin
Actinomycin D; Noncoding RNAs; Pulse labeling with uridine analogs; RNA half-life; RNA stability; Transcription inhibitors