Circularized RT-PCR (cRT-PCR): analysis of the 5' ends, 3' ends, and poly(A) tails of RNA

Methods Enzymol. 2013;530:227-51. doi: 10.1016/B978-0-12-420037-1.00013-0.

Abstract

Many techniques that are used to characterize individual RNA molecules can potentially alter the original transcript sequence or its posttranscriptional modifications, such as polyadenylation. Methods that are designed to define the ends of an RNA molecule, for example, oligonucleotide ligation, avoid altering the transcript sequence but can usually fulfill only one objective per experiment (e.g., define the 5' or the 3' end). In contrast, not only does circularized reverse transcription coupled with PCR (cRT-PCR) preserve the original 5' and 3' ends of the transcript and posttranscriptionally added extensions, but also the material from one experimental procedure can be utilized in order to characterize both the 5' and 3' ends. Furthermore, if suitable oligonucleotide primers are designed, cRT-PCR can be used to isolate truncated, adenylated (and nonadenylated) molecules that are intermediates in RNA decay (Slomovic and Schuster, 2008).

Keywords: Circularized RT-PCR (cRT-PCR); Electroporate and plate bacteria; Gel purification; Gene-specific reverse primer; PCR products; Plasmid DNA isolation and sequencing; RNA purification.

MeSH terms

  • Bacteria / chemistry
  • Bacteria / genetics
  • Base Sequence
  • DNA / chemistry
  • DNA / genetics
  • DNA / isolation & purification
  • Electrophoresis, Polyacrylamide Gel / methods
  • Electroporation / methods
  • Plasmids / chemistry
  • Plasmids / genetics
  • Plasmids / isolation & purification
  • Poly A / analysis*
  • Polyadenylation
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcription
  • Sequence Analysis, DNA / methods

Substances

  • RNA, Messenger
  • Poly A
  • DNA