Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence

Michael J. Fasco, Chris P. Treanor, Simon Spivack, Helen L. Figge, Laurence S. Kaminsky

Research output: Contribution to journalArticle

53 Scopus citations

Abstract

Quantitative RNA-polymerase chain reaction (RNA-PCR) is an extremely powerful analytical tool owing to its specificity and high level of sensitivity. Quantitative RNA-PCR is, however, highly labor intensive. No analytical method currently exists that can accurately and rapidly quantitate the small quantities of DNA in RNA-PCR reaction mixtures. We have developed a method using capillary electrophoresis and laser-induced fluorescence to detect YOYO-1 complexes of DNA produced by PCR. RNA-PCR mixtures can be analyzed either directly (without primer and protein removal) or by electrokinetic injection following desalting. Modified competitive and multiplex competitive RNA-PCR assays for glyceraldehyde-3-phosphate dehydrogenase and P4501A1 were tested in a series of mixtures containing equal concentrations, but different proportions, of RNA from untreated (essentially no P4501A1 mRNA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated (high levels of P4501A1 mRNA) HepG2 cells. Twofold differences in concentrations between two P4501A1 mRNA solutions could be detected by competitive RNA-PCR. Glyceraldehyde-3-phosphate dehydrogenase concentrations were constant throughout. Multiplex competitive PCR produced more variable results due to the presence of contaminating peaks, which hindered accurate area integration. These data demonstrate the potential usefulness of capillary electrophoresis in a variety of quantitative PCR applications.

Original languageEnglish (US)
Pages (from-to)140-147
Number of pages8
JournalAnalytical Biochemistry
Volume224
Issue number1
DOIs
Publication statusPublished - Jan 1995
Externally publishedYes

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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