Quantitative analysis of gene expression in different tissues by template-calibrated RT-PCR and laser-induced fluorescence.
RT-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of, at times complex, procedures, quantitation of RT-PCR remains difficult, particularly when comparing RNA from different tissues or very small samples. In the procedure described here, we calibrate input cDNA through incorporation of trace label. PCR product is generated from equal amounts of cDNA with fluoresceinated primers, size fractionated, and quantitated by laser-induced fluorescence in an automated DNA sequencer. Eliminating variation in input cDNA resulted in reliable noncompetitive PCR quantitation from templates equivalent to > or = 50 pg of total RNA. Using the example of beta-glucuronidase, a low-copy-number housekeeping gene, we have drawn a map of differential gene expression for this protein in various rat tissues.
Karges
W J
WJ
Department of Pediatrics and Immunology, Hospital for Sick Children, Toronto, Canada.
Gaedigk
R
R
Dosch
H M
HM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D015870:Gene Expression; D005966:Glucuronidase / Q000032:analysis / Q000096:biosynthesis*; D007834:Lasers; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence / Q000379:methods; D013698:Templates, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D005966:Glucuronidase