Quantitative Measurement of Relative Gene Expression in Human Tumors

Abstract

Quantitative measurement of specific mRNA species is of major importance for approaching many fundamental questions in biology. Until now, quantitation of gene expression has usually been done by Northern blotting, but this procedure is relatively insensitive, requiring microgram amounts of RNA. Furthermore, unless linear ranges of RNA concentration are determined, the procedure is semiquantitative at best. Because of the limitations of Northern blotting, various strategies have been developed for quantitation of cDNA by polymerase chain reaction (PCR)-based methods (1-3), most of them utilizing the principle of competitive PCR in which a synthetic segment is coamplified along the target DNA segment. We were interested in comparing the expression of drug target genes in very small samples of human tumor material, such as would be obtained from biopsies or even paraffin blocks. The competitive PCR was not entirely suitable for this purpose because: (1) the "input" RNA or DNA concentration must be known with precision in order to provide a normalization factor, and (2) our results suggested that the competitor and target were not necessarily amplified with the same efficiency even when the segments to be amplified were the same size (4). To overcome these problems, we developed an alternate method of PCR quantitation with the following key features:

Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation

“…The gels were subsequently quantified with the IPLab Images software as well as using a PhosphorImager and the ImageQuant software (Molecular Dynamics). The relative levels of gene expression were measured by determining a ratio between the products generated from the target gene and the endogenous internal standard in separate reactions (35). The linear range for amplifying regions of the genes of interest and the gene for the internal standard were determined by serial dilutions of the cDNA.…”

Multidrug Resistance in Ovarian Cancer: Comparing an Immunocytochemical Study and ATP-Tumor Chemosensitivity Assay

“…The possibility of optimizing anticancer chemotherapy is today provided when studying the chemosensitivity or resistance of tumor explants from cancer patients using innovative, highly reliable, biochemical, molecular or immunohistochemical methodologies, capable of detecting the expression of gene products responsible for the occurrence of these phenomena 33 , 34 and by use of in vitro chemosensitivity tests 12,13,14,16,35 . In the near future, data obtained from these methods may contribute to the prediction of clinical response to specific antineoplastic agents, and help guide the choice of optimum pharmacological treatment copy for private use only for individual patients.…”

Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells

“…Therefore, a relatively small reduction in the amount of DNA-PK cs mRNA could prevent detection in M059J cells by this method. The expression of a gene can be quantitatively compared between cell lines or cell types from standard curves produced by simultaneous PCR on serial dilutions of cDNA reactions (Horikoshi et al, 1992). At approximately high dilutions, a linear relationship exists between the template dilution and the amount of PCR product, but at lower dilutions, when the concentration of cDNA template corresponding to the mRNA species of interest is ample, the reaction saturates.…”

“…Several other sequencing primers were used which are internal to the PCR products generated by PK23 and PK27 or PK5 and PK7. The bactin primers (BA-67 and BA-68) are described by Horikoshi et al (1992).…”