How is StaRT-PCR™ different from Real-time RT-PCR?
The fundamental differences between StaRT-PCR™ and kinetic (real-time RT-PCR) technology gene expression measurement values are two-fold. First, StaRT-PCR employs standardized mixtures of internal standards (SMIS™) all of which include a internal standard for a reference gene. Second, data are obtained in different phases of the PCR cycle. StaRT-PCR™ analyses occur at the plateau phase (end-point) and measure the gene of interest and a reference gene (i.e. ϐ-actin, ACTB) relative to its corresponding internal standard. Then, the expression of each target gene relative to the reference gene is compared to obtain a numerical value in units of molecules of target gene / 106 ACTB molecules (1).

Real-time PCR analyses occur at the log-linear phase and the template quantity value is calculated from the threshold cycle (CT), the point at which the fluorescence passes from insignificant levels to detectable levels. Titrations of an external standard allows for the construction of a standard curve permitting the concentration of unknown samples to be determined (2-6).

Why Utilize Standardized Mixtures of Internal Standards in Quantitative RT-PCR?
There is an urgent need to eliminate the variability of quantitative RT-PCR results obtained by the same samples assayed in different laboratories (7). The cause of the discrepancy is the worker and the reagents. The solution is to utilize standardized mixtures of internal standards to control for the sources of variation. Table 1 outlines several sources of variation in quantitative RT-PCR and the method to control for them.

Both StaRT-PCR™ and kinetic RT-PCR technologies can correct for cDNA loading due to pipetting, quantification, or reverse transcription by multiplexing with a reference gene (8). Similarly, both technologies can control for the efficiency of amplification due to cycle-to-cycle and gene-to-gene variation (9-11). However, the only way to control for variation in PCR reaction efficiency due to inhibitors present in samples (sample-to-sample variation) (12), in the quality of lot prepared PCR reagents (reaction-to-reaction variation), or in location the tube is placed within the thermocycler (position-to-position variation) is to include an internal control (13). In addition, another benefit of utilizing standardized mixtures of internal standards is that quantification may be determined at end point, eliminating the need for real-time analysis (14).

How can I place an order for the StaRT-PCR™ assay service?
Each order is customized to the individual client. Gene Express Inc. has several hundred StaRT-PCR™ assays available for immediate use. A list of genes can be found at www.GeneExpressInc.com. For more information concerning discount pricing and turnaround times, please contact:

Gene Express, Inc.
1410 Commonwealth Dr., Suite 105
Wilmington, NC 28403

Phone: 800-820-8341
Fax: 800-820-8343
email: info@GeneExpressInc.com

www.GeneExpressInc.com

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