Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.

Original languageEnglish (US)
Pages (from-to)524-531
Number of pages8
JournalBioTechniques
Volume28
Issue number3
StatePublished - 2000

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RNA
Polymerase Chain Reaction
Messenger RNA
Transcription
Reverse Transcription
Complementary DNA
RNA Cleavage
Degradation
Molecules
Population
Amplification
Assays
Tissue
oligo (dT)

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA. / Swift, Galvin H.; Peyton, Michael J.; MacDonald, Raymond J.

In: BioTechniques, Vol. 28, No. 3, 2000, p. 524-531.

Research output: Contribution to journalArticle

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