Standardized Mixtures of Internal Standards™ (SMIS™) Provide for Standardized Live Data

The measurement of gene expression patterns is rapidly becoming the method of choice to determine molecular signatures indicative of disease. The gold standard for gene expression measurement is competitive PCR. The reasons for this are many. The method is highly sensitive and it can measure gene expression over the entire range of expression levels observed in cells. Perhaps the most important benefit of competitive PCR is the inclusion of internal standards that corrects for any aberrations in efficiency between PCR reaction vessels. However, until recently, competitive PCR has not been widely employed for measurement of gene expression because of the labor-intensive process of constructing competitive templates that are used as the internal standards. In addition, without the availability of Standardized Mixtures of Internal Standards™ (SMIS™), the method still has the potential to yield variable results from assay to assay. As the result of focused, intensive research, Gene Express, Inc. has solved many of the problems previously associated with competitive PCR. We manufacture proprietary SMIS™ for hundreds of genes and offer a gene expression analysis service employng these SMIS™ to researchers. These factors provide researchers the opportunity to generate standardized "live" data. We call our technology StaRT-PCR™, for Standardized, Reverse Transcriptase PCR.

The Introduction of StaRT-PCR™
StaRT-PCR™ was first described in 1998 by Dr. James Willey, the inventor of the technology and scientific and medical consultant to Gene Express, Inc. (1). In this paper, Dr. Willey described the importance of using standardized mixtures of internal standards in generating valid and reproducible gene expression data for multiple genes.

Multiple Laboratory Validation of StaRT-PCR™ Shows High Reproducibility
A method that provides standardized, reproducible gene expression data is a prerequisite to the development of a meaningful database suitable for conducting multi-institutional clinical studies based on expression measurement and applicable to the development of rigorous diagnostic tests. As discussed above, StaRT-PCR™ has all these characteristics. In addition, any method used for this purpose must have high reproducibility. To address this issue, a multi-laboratory study to evaluate both the intralaboratory and interlaboratory reproducibility of StaRT-PCR™ has been performed and published (2). In a blinded study, the expression of ten genes was measured by StaRT-PCR™ in a cDNA sample provided to each of four laboratories by Gene Express, Inc.

The results of that study document the high reproducibility that can be obtained with StaRT-PCR™. It should be noted that those results were obtained using conventional hand-held pipettes with no controlling for electrophoretic methods. Since this publication, we have shown that CVs with our newly implemented, high throughput robotics system and using the Caliper Life Scieces AMS-90™ micorcapillary suystem, in the tightly controlled environmant of the SEM™ Center are even lower (<12%).

Standardized Numerical Values Allow for Calculation of Interactive Gene Expression Indices
It is likely that most phenotypes will be associated with a particular pattern of expression of more than one gene. StaRT-PCR™ provides data that can be mathematically presented as interactive gene expression indices (IGEIs). Typically, positively associated gene expression values are in the numerator and negatively associated values in the denominator. IGEIs have been identified for bronchogenic carcinoma (3) and anti-folate resistance among childhood leukemia (4, 5).

Independent Validation of StaRT-PCR™
StaRT-PCR™ has also been used to evaluate detoxification in keratinopcytes (6) the CFTR gene in bronchial epithelial cells (7), cytokine genes in bronchial epithelial cells (8), and xenobiotic metabolism enzyme genes in bronchial epithelial cells (9). There are currently many other ongoing projects utilizing StaRT-PCR™.

1. Willey J.C., Crawford E.L., Jackson C.M. Expression measurement of many genes simultaneously by quantitative RT-PCR using standardized mixtures of competitive templates. Am. J. Respir. Cell Mol. Biol., 19: 6-17, 1998.
2. Crawford E.L., Peters G.J., Noordhuis P., et al. Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR. Mol. Diagn., 6: 217-225, 2001.
3. DeMuth J.P., Jackson C.M., Weaver D.A., et al. The gene expression index c-myc x E2F1/p21 is highly predictive of malignant phenotype in human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol., 19: 18-24, 1998.
4. Rots, M.G., Willey, J.C., Jansen, G., et al. mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method. Leukemia, 14: 2166-2175, 2000.
5. Rots, M.G., Pieters, R., Peters, G.J., et al. Circumvention of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates. Blood, 94: 3121-3128, 1999.
6. Vondracek M.T., Weaver D.A., Sarang Z., et al. Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and Simian virus 40 T antigen-immortalized oral keratinocytes. Int. J. Cancer, 99: 776-782, 2002.
7. Allen J.T., Knight R.A., Bloor C.A., Spiteri M.A. Enhanced insulin-like growth factor binding protein-related protein 2 (connective tissue growth factor) expression in patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis. Am. J. Respir. Cell. Mol. Biol., 21: 693-700, 1999.
8. Loitsch S.M., Kippenberger S., Dauletbaev N., Wagner T.O., Bargon J. Reverse transcription-competitive multiplex PCR improves quantification of mRNA in clinical samples- application to the low abundance CFTR mRNA. Clinical Chem., 45: 619-624, 1999.
9. Mollerup S., Ryberg D., Hewer A., Phillips D.H., Haugen A. Sex differences in lung CYP1A1 expression and DNA adduct levels among lung cancer patients. Cancer Res., 59: 3317-3320, 1999.

Other Relevant StaRT-PCR™ References
Crawford EL, Warner KA, Khuder SA, Zahorchak RJ, & Willey JC. 2002. Multiplex standardized RT-PCR for expression analysis of many genes in small samples. Biochem Biophys Res Commun. 293:509-516.

Apostolakos MJ, Schuermann WH, Frampton MW, Utell MJ, & Willey JC. 1993. Measurement of gene expression by multiplex competitive polymerase chain reaction. Anal Biochem. 213:277-284.

Hermess, EA, & Naz, RK. 2003. A novel Human Prostate-specific Gene-1 (HPG-1): Molecular cloning, sequencing, and its potential involvement in prostate carcinogenesis. Cancer Research. 63:329-336.

Lechner JF, Wang Y, Siddiq F, Fugaro JM, Wali A, Lonardo F, Willey JC, Harris CC, & Pass HI. 2002. Human lung cancer cells and tissues partially recapitulate the homeobox gene expression profile of embryonic lung. Lung Cancer. 37:41-7.

Crawford, E.L., D.A. Weaver, J.P. demuth, C.M. Jackson, S.A. Khuder, M.W. Frampton, M.J. Utell, W.G. Thilley, & J.C. Willey. 1998. Measurement of cytrochrome P450 2A6 and 2E1 gene expression in primary human bronchial epithelial cells. Carcinogenesis. 19:1867-1871.