EXPRESSION MEASUREMENT OF MANY GENES SIMULTANEOUSLY BY QUANTITATIVE RT-PCR USING STANDARDIZED MIXTURES OF COMPETITIVE TEMPLATES

Progress toward complete sequencing of all human genes through the Hum an Genome Project has already resulted in a need for methods that allo w quantitative expression measurement of multiple genes simultaneously . It is increasingly recognized that relative measurement of multiple genes will provide more mechanistic information regarding cell pathoph ysiology than measurement of individual genes one by one or by methods that do not allow direct intergene comparison. In this study, previou sly described quantitative reverse transcription-polymerase chain reac tion methods were modified in an effort to provide a rapid, simple met hod for this purpose. Internal standard competitive templates (CTs) we re prepared for each gene and were combined in a single solution conta ining CTs for more than 40 genes at defined concentrations relative to one another. Any subsequent dilution of the CT mixture did not alter the relationship of one CT to another. Because the same CT standard so lution or a dilution of it was used in all experiments, data obtained from different experiments were easily compared. The use of multiple C T mixtures with different housekeeping gene to target gene ratios prov ided a linear dynamic range spanning the range of expression of an gen es thus far evaluated. CT stock solutions were used to simultaneously quantify the expression of 25 genes relative to beta-actin and glycera ldehyde-3-phosphate dehydrogenase in normal and malignant bronchial ep ithelial cells. Because the CT concentrations were known, data in the form of both absolute messenger RNA (mRNA) copy number and mRNA relati ve to housekeeping gene mRNA were obtained. The methods and reagents d escribed will allow rapid, quantitative measurement of multiple genes simultaneously, using inexpensive and widely available equipment. Furt hermore, the CT standard solution may be distributed to other investig ators for interlaboratory standardization of experimental conditions.