Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression.
The ability of the polymerase chain reaction (PCR) to quantitate expression of mRNA was examined in the present study. The model chosen was expression of the multidrug resistance gene mdr-1/Pgp in two colon carcinoma cell lines which express mdr-1/Pgp at levels comparable to those found in many clinical samples. PCR was utilized to evaluate differences in mdr-1/Pgp expression in the two cell lines after modulation by sodium butyrate. Thus, comparisons were made across a range of mdr-1/Pgp expression as well as comparisons of small differences. The PCR was found to be both sensitive and quantitative. Accurate quantitation requires demonstration of an exponential range which varies among samples. The exponential range can be determined by carrying out the PCR for a fixed number of cycles on serial dilutions of the RNA reverse transcription product, or by performing the reaction with a varying number of cycles on a fixed quantity of cDNA. By quantitation of the difference in PCR product derived from a given amount of RNA from the sodium butyrate treated and untreated cells, the difference in mRNA expression between samples can be determined. Normalization of the results can be achieved by independent amplification of a control gene, such as beta 2-microglobulin, when the latter is also evaluated in the exponential range. Simultaneous amplification of the control and target genes results in lower levels of PCR products due to competition, which varies from sample to sample. The PCR is thus a labor-intensive but sensitive method of quantitating gene expression in small samples of RNA.
Murphy
L D
LD
Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.
Herzog
C E
CE
Rudick
J B
JB
Fojo
A T
AT
Bates
S E
SE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D004247:DNA / Q000096:biosynthesis; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002087:Butyrates; D003845:Deoxycytosine Nucleotides; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D020148:Butyric Acid; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA