All of our StaRT-PCR™ assays are performed under strictly controlled GLP (Good Laboratory Practice) conditions employing our proprietary Standardized Mixtures of Internal StandardsTM (SMISTM) and gene-specific primers in our Standardized Expression Measurement (SEM) Center™. The SEM Center™ uses high throughput, automated equipment and proprietary software that enables file-based experimentation ensuring all assays are setup, performed, and analyzed with little human intervention. All assays are performed under the FDA Good Laboratory Practice (GLP) regulations (21 CFR Part 58) and in compliance with Gene Express' defined and tested SOPs (Standardized Operation Procedures). The SMIS™ contain competitive Internal Standards at precisely known quantities for each transcript to be measured and are added to the cDNA sample prior to aliquotting for PCR reaction. Gene-specific primers are added to the cDNA and SMIS™ mixture and amplified by PCR. "StaRT-PCR™" - Standardized (Sta), Reverse Transcriptase (RT), Polymerase Chain Reaction (PCR) - PCR is an enzymatic process to increase the number of copies of DNA for easier detection. A microfluidic capillary electrophoresis instrument (Caliper LabChip 90 Electrophoresis System) is used to accomplish high throughput, automatic gene expression analysis. The amount of transcript for each gene in a sample is measured relative to its respective Internal Standard within the Standardized Mixture of Internal Standards™ (SMIS™). This is done by electrophoretically separating the PCR products in a microcapillary channel in the presence of a fluorescent intercalater dye. The PCR product peak area for each transcript is then compared to the peak area of the PCR product for the respective Internal Standard. The Internal Standard controls not only for inter-sample and inter-experimental variation in PCR amplification efficiency, but also for variation in sample loading and pipetting of PCR product into the analytic device. Also, note that there is no requirement for incorporation of expensive sequence-specific, fluorescently-labeled probes.

The SMIS™ contain competitive Internal Standards at precisely known quantities for each transcript to be measured and are added to the cDNA sample. Gene-specific primers are added to the cDNA and SMIS™ mixture and amplified by PCR. The amount of transcript for each gene in a sample is measured relative to its respective Internal Standard within the Standardized Mixture of Internal Standards™ (SMIS™). These mixtures of Internal Standards allow for the simple calculation (no time consuming statistical analysis is needed) of numbers of molecules of transcripts for the target gene relative to a reference gene and to each other gene measured. All data are reported as a number of mRNA molecules for gene "X" per 106 molecules of reference gene. If desired, these standardized numeric gene expression values can be easily normalized to any transcript that is measured through simple mathematical calculations. Since numerical values are determined, data from different samples and different experiments can be directly compared.

StaRT-PCR™ reagents for each gene (gene-specific primers and Internal Standard) are checked for transcript specificity both in-silico and empirically. Additional gene-specificity for each analysis is provided because the expected size of the transcript native template and its Internal Standard are known and verified with our proprietary software.

RNA Quantity Needed for StaRT-PCR™ Assay

The amount of RNA to reverse transcribe to cDNA varies from 100 ng to 100 pg. This depends on the number of genes to be tested per sample and the level of expression of the genes.

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