Transcript quantitation in total yeast cellular RNA using kinetic PCR.
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.
Kang
J J
JJ
Department of Biological Chemistry, School of Medicine, University of California, 1 Shields Avenue, Davis, CA 95616, USA.
Watson
R M
RM
Fisher
M E
ME
Higuchi
R
R
Gelfand
D H
DH
Holland
M J
MJ
D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D005800:Genes, Fungal; D009154:Mutation; D012331:RNA, Fungal / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics
D017931:DNA Primers; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger