Expression Measurement of Many Genes Simultaneously by Quantitative RT-PCR Using Standardized Mixtures of Competitive Templates

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Abstract
Progress toward complete sequencing of all human genes through the Human Genome Project has already resulted in a need for methods that allow quantitative expression measurement of multiple genes simultaneously. It is increasingly recognized that relative measurement of multiple genes will provide more mechanistic information regarding cell pathophysiology than measurement of individual genes one by one or by methods that do not allow direct intergene comparison. In this study, previously described quantitative reverse transcription-polymerase chain reaction methods were modified in an effort to provide a rapid, simple method for this purpose. Internal standard competitive templates (CTs) were prepared for each gene and were combined in a single solution containing 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 solution or a dilution of it was used in all experiments, data obtained from different experiments were easily compared. The use of multiple CT mixtures with different housekeeping gene to target gene ratios provided a linear dynamic range spanning the range of expression of all genes thus far evaluated. CT stock solutions were used to simultaneously quantify the expression of 25 genes relative to beta-actin and glyceraldehyde-3-phosphate dehydrogenase in normal and malignant bronchial epithelial cells. Because the CT concentrations were known, data in the form of both absolute messenger RNA (mRNA) copy number and mRNA relative to housekeeping gene mRNA were obtained. The methods and reagents described will allow rapid, quantitative measurement of multiple genes simultaneously, using inexpensive and widely available equipment. Furthermore, the CT standard solution may be distributed to other investigators for interlaboratory standardization of experimental conditions.