Use of the polymerase chain reaction in the quantitation of MDR-1 gene expression

Abstract

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.

Sensitivity to CPT-11 of xenografted human colorectal cancers as a function of microsatellite instability and p53 status

“…CRC xenografts displayed a large range of sensitivity to CPT-11, from resistance to eradication. These differences were not due to different levels of top1, as evaluated by a semiquantitative method (Murphy et al, 1990), as already published (Goldwasser et al, 1995;van Ark-Otte et al, 1998). Jansen et al (1998) proposes a role for P-glycoprotein (encoded by mdr1 gene) in lack of efficacy of numerous compounds, including CPT-11.…”

Paclitaxel-resistant Human Ovarian Cancer Cells Have Mutant β-Tubulins That Exhibit Impaired Paclitaxel-driven Polymerization

“…Quantitative PCR-Quantitative PCR for ␤-tubulin isotypes was performed as described previously (24). Specific primers for each ␤-tubulin isotype: M40 (class I), ␤9 (class II), ␤4 (class III), 5␤ (class IVa), ␤2 (class IVb), were utilized as described previously (16) (arabic numerals refer to the human gene, and roman numerals refer to the protein isotype class (25)).…”

“…These include reduced total tubulin levels (10,24), increased tubulin content (15), differential migration of ␣-or ␤-tubulin on two-dimensional polyacrylamide gel electrophoresis (11,13), acetylation of ␣-tubulin (18), and increased expression of specific tubulin isotypes (14 -17). In the present study, the total tubulin content of the resistant cells was similar to that of the parental cells (data not shown), and no differences in the acetylation of purified ␣-tubulin were observed (data not shown).…”

Cytoskeleton and paclitaxel sensitivity in breast cancer: The role of β‐tubulins

“…4,15 Specific primers for each isotype have been already described. 16 The cDNA was serially diluted from 500 to 0.015 ng of RNA and PCR reactions conducted as previously reported.…”