Precision Profiles™

Source MDx has developed a molecular diagnostic assay (Precision Profile™) based on RNA transcript measurement using quantitative RT-PCR optimized for precision and calibration (Source MDx US patent 6692916B2). The assay is robust in its measurement of a defined panel of target genes (Precision Profile™) used to evaluate a biological condition or the effect of an agent in human or animal samples derived from bodily fluids, tissues or culture medium. Total RNA is extracted from each sample and a first strand synthesis is performed using a reverse transcriptase (RT) to generate cDNA. The cDNA serves as a template for use in the Precision Profile™ assay where individual target gene amplification is assayed by quantitative RT-PCR using the ABI Prism® 7900HT Sequence Detection System. Precision Profile™ assays are transferable to alternative PCR platforms as part of Source MDx's platform independent approach in the development of molecular diagnostic tests.

Key to the Precision Profile™ assay is the concept of precision (repeatability and reproducibility) and calibration (closely matched amplification efficiencies) achieved through proprietary reagents (target gene primer probe sets), high performance 384-well assay plates and strict adherence to narrow permissible levels of experimental variables. By rigorously controlling all variables associated with the assay, starting from sample collection and processing, key reagent preparation and qualification to instrument runs and data analysis, the gene expression levels in a sample of a given biological condition can be measured and clear comparisons can be made between gene loci, biological conditions and individuals. All Source MDx processes from sample collection to data analysis are performed according to Source MDx established Standard Operating Procedures (SOPs).

The technical validation of any set of target genes intended for use in a Precision Profile™ assay, particularly in a clinical validation setting, is of paramount importance. At Source MDx technical validation focuses on the quality and performance of reagents (primer probe sets, endogenous controls and Master Mix), which will be used in a defined process to provide a reliable measurement of gene expression for use in a clinical setting. Source MDx technically validates newly designed target gene primer probe sets and further qualifies their performance at the production scale level and plate production level in three consecutive phases:

• Design Scale Primer Probe Quality Control
  Individual target gene primer probe sets are optimized through multiple version design and testing. Primer probe design candidates undergo quality control testing to ensure that target specificity, amplification and efficiency are within required specifications. Having met all the acceptance criteria, a single optimal primer probe set is selected for use as a reagent at the production scale.
• Production Scale Primer Probe Quality Control
  Production scale primer probe reagents are also required to undergo quality control testing in order to ensure that the production scale lot is performing to the specifications established at the design scale. All future primer probe production scale lots are quality control tested by comparison to previous production scale lots. Source MDx's in-house primer probe database tracks all design and quality control performance metrics, ensuring consistent and traceable reagent quality and performance over time.
• Precision Profile™ Plate Production and Quality Control
  Precision Profile™ plates are configured in a 384-well format consisting of triplicate wells of individual target gene primer probe sets multiplexed with an endogenous control and Master Mix. All Precision Profile™ plates adhere to a quarantine (prior to QC testing) and release (post QC testing) process, after which only automated template addition is required.

Precision Profile™ plate production runs are prepared in batches of up to 150 plates using pre-qualified reagents and liquid handling automation. Precision Profile™ plate performance is further qualified in test plates selected from the production batch by PCR analysis using a Source MDx standard cDNA template. Production batches must meet quality control metrics as defined by comparability of test plate data to previously established baseline data (using a common standard cDNA template) prior to release. This process ensures consistent and reliable Precision Profile™ plate performance over time and across subject samples. Precision Profile™ plate production runs over time typically yield coefficients of variance (CVs) of less than 1% for most target genes (based on target gene measurements using Source MDx standard cDNA template).