Overview of StaRT-PCR™
StaRT-PCR™ (Standardized Reverse Transcriptase 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 competitive templates as internal standards (CT) in generating valid and reproducible numerical gene expression data for multiple genes. Based on quantitative, competitive PCR (qcPCR), StaRT-PCR™ is a patented, revolutionary improvement over conventional qcPCR and other methods. Gene Express has licensed the technology to provide a service that provides numerical, standardized gene expression values for our customers. Figure 1 is a diagrammatic representation of the StaRT-PCR™ process as performed in our Standardized Expression Measurement (SEM) Center™.

RNA extraction and reverse transcription. As with most gene expression measurement methods, total RNA must be extracted either from cells grown in vitro or from tissue samples. Gene Express provides this service. Tissue samples must be snap-frozen immediately in liquid nitrogen upon procurement to avoid degradation or changes in the relative amount of the various transcripts present. Figure 1 depicts transcripts for three genes expressed at three different levels, blue [low], orange [medium], and purple [high]). If the tissue or cells are harvested and subsequently handled and extracted correctly, the ratios of the transcripts for these three genes in the total RNA will reflect that in the cell. Once reverse transcribed, the resulting cDNA will contain the same ratios of gene-specific cDNA. These concepts apply to virtually all gene expression measurement technologies. It is in the following steps where StaRT-PCR™ provides added value over other gene expression measurement methods.

Fig. 1. Diagram of the StaRT-PCR™

Preparing the StaRT-PCR™ master mix. Once the RNA is converted to cDNA, the cDNA is mixed with our proprietary internal standard mixture of CTs. In our standard mixture, there is an internal standard CT for each gene to be measured as well as one for a reference gene (i.e., ACTB, GAPD, etc.). It is very important to note that because our standardized mixtures are prepared in bulk (enough for trillions of assays) and because they are added at this time, the ratio of each internal standard CT to each other CT and each internal standard CT to its native transcript (NT) is fixed. This is critical for obtaining standardized data that can readily be cross-referenced among samples and laboratories. This also allows for control of loading in later steps as well as for any differences in PCR amplification between samples, reactions, cycler location, etc.

Performing the PCR reactions. Once the master mix is made, fixing all CT/NT ratios, PCR buffer and Taq polymerase are added to the mixture. After aliquotting the mixture into wells of a 96- or 384-well plate. One gene-specific primer pair is then added to each well. In Figure 1 the primer pair for the blue gene is placed in the first tube, the primer pair for the orange gene in the second tube and the primer pair for the purple gene in third tube. Note that, except for the primer pairs, all tubes contain the same reagents in the same concentrations. The PCR amplification is then done. During the amplification, each gene-specific primer pair amplifies its specific NT and CT with the same kinetics because the priming sequences are identical and ample time is allowed during the extension step to allow complete polymerization at each cycle.

Analyzing StaRT-PCR™ products. The amplicons produced by StaRT-PCR™ can be analyzed by any currently available electrophoretic method. Figure 1 depicts separation on agarose gel (lower left) or capillary electrophoresis (CE) (lower right). Our Standardized Expression Measurement (SEM) Center™ currently uses a Caliper AMS-90 microfluidic capillary electrophoresis instrument to accomplish, high-throughput automatic analysis. The amount of CT or NT amplimer is determined by either measuring band density (agarose) or peak area (CE). Note that there is no requirement for incorporation of expensive fluors or quenchers to detect the amplimers. Additional specificity control for each is provided because the expected size of the CT and NT amplicons is known and verified. Our proprietary software verifies the size for each expected product. All data are then reported as number of molecules mRNA for gene "X" per 106 molecules of ACTB (reference gene). Serial dilutions of the standardized mixes allow quantitative measurements over the entire 6.5 log range of gene expression. Order today, StaRT-PCR™ assays for multi-gene expression analysis.

Relevant References

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.