RT-qPCR Detection of Senescence-Associated Circular RNAs.
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.
Laboratory of Genetics and Genomics, National Institute on Aging-Intramural Research Program, National Institutes of Health, 251 Bayview Blvd., Baltimore, MD, 21224, USA.
Amaresh C
AC
Panda
Laboratory of Genetics and Genomics, National Institute on Aging-Intramural Research Program, National Institutes of Health, 251 Bayview Blvd., Baltimore, MD, 21224, USA. abdelmohsenk@mail.nih.gov.
Kotb
K
Abdelmohsen
Laboratory of Genetics and Genomics, National Institute on Aging-Intramural Research Program, National Institutes of Health, 251 Bayview Blvd., Baltimore, MD, 21224, USA.
Myriam
M
Gorospe
D016922:Cellular Senescence / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D000079962:RNA, Circular; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012984:Software; D057188:Workflow
D000079962:RNA, Circular; D012313:RNA
Divergent primer design; RNA-binding proteins; Sponge circRNAs; Transcriptome