Transcript quantitation in total yeast cellular RNA using kinetic PCR

Abstract

Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT-PCR, kRT-PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT-PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of +/-20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators.

Figure 1

(A) Photograph of the kinetic thermal cycler (KTC). The optical components of the KTC are contained within a light-tight enclosure with a door (open) positioned over the sample compartment of a Perkin Elmer 9600 thermal cycler. A 300 nm UV lamp shines into the enclosure through a dichroic mirror onto the sample block. Fluorescence from the samples is reflected by the dichroic mirror to the rear of the enclosure, through a Fresnel lens and interference filter onto a CCD video camera. (B) Schematic of the arrangement of the KTC optical components.

Figure 2

Kinetic RT–PCR curves for the PGK1-D primer pair and total yeast RNA template. Relative fluorescence output is plotted versus PCR cycle number. Kinetic curves are for 10⁵ (triangles), 10⁴ (diamonds) and 10³ (squares) cell equivalents of total yeast cellular RNA template (23) and no added template (open circles).

Figure 3

Kinetic RT–PCR analysis of transcripts from the yeast GAL1/GAL10 intergenic region. Relative fluorescence output is plotted versus PCR cycle number. Kinetic RT–PCR assays were performed with 10⁵ cell equivalents (0.12 µg) of total yeast cellular RNA isolated from a wild-type strain grown in YPd medium. Kinetic curves are shown for the GAL1/GAL10 primer pair using: untreated RNA template (triangles); RNA template pretreated with RNase A (squares); RNA template pretreated with RNase A and DNase I (open circles). The kRT–PCR curve for the TDH3 primer pair (crosses) using 10⁵ cell equivalents (0.12 µg) of total yeast cellular RNA isolated from a wild-type strain grown in YPd medium is also shown.