RT-qPCR Detection of Senescence-Associated Circular RNAs

Methods Mol Biol. 2017;1534:79-87. doi: 10.1007/978-1-4939-6670-7_7.

Abstract

Primary cells that reach the end of their replicative potential, encounter sublethal stress, or experience the activation of certain oncogenes cease proliferation and enter a state of long-term growth arrest named senescence. The senescent process has been implicated in a variety of age-related diseases and also in the pathogenesis of cancer. Senescence is characterized by distinct changes in the types and levels of coding RNAs (mRNAs) as well as in the vast collective of regulatory noncoding (nc)RNAs, which includes microRNAs, long noncoding RNAs (lncRNAs), and circular (circRNAs). Numerous technologies permit the detection of senescence-associated linear transcripts (mRNAs, lncRNAs, microRNAs), but the identification and quantification of circRNAs in senescence require distinct molecular approaches. In this chapter, we describe a method for the detection and measurement of circRNAs in senescent cells using specialized reverse transcription (RT) followed by real-time quantitative (q)PCR analysis.

Keywords: Divergent primer design; RNA-binding proteins; Sponge circRNAs; Transcriptome.

MeSH terms

  • Cellular Senescence / genetics*
  • Computational Biology / methods
  • RNA / genetics*
  • RNA / isolation & purification
  • RNA, Circular
  • Real-Time Polymerase Chain Reaction* / methods
  • Reproducibility of Results
  • Sequence Analysis, DNA
  • Software
  • Workflow

Substances

  • RNA, Circular
  • RNA