Analytical validation of the Oncotype DX prostate cancer assay - a clinical RT-PCR assay optimized for prostate needle biopsies.

BACKGROUND: The Oncotype DX Prostate Cancer Assay is a multi-gene RT-PCR expression assay that was developed for use with fixed paraffin-embedded (FPE) diagnostic prostate needle biopsies containing as little as 1 mm of prostate tumor in the greatest dimension. The assay measures expression of 12 cancer-related genes representing four biological pathways and 5 reference genes which are algorithmically combined to calculate the Genomic Prostate Score (GPS). This biopsy-based assay has been analytically and subsequently clinically validated as a predictor of aggressive prostate cancer. The aim of this study was to validate the analytical performance of the Oncotype DX Prostate Cancer Assay using predefined acceptance criteria. RESULTS: The lowest quartile of RNA yields from prostate needle biopsies (six 5 µm sections) was between 19 and 34 ng. Analytical validation of the process requiring as little as 5 ng of RNA met all pre-defined acceptance criteria. Amplification efficiencies, analytical sensitivity, and accuracy of gene assays were measured by serially diluting an RNA sample and analyzing features of the linear regression between RNA expression measured by the crossing point (Cp) versus the log2 of the RNA input per PCR assay well. Gene assays were shown to accurately measure expression over a wide range of inputs (from as low as 0.005 ng to 320 ng). Analytical accuracy was excellent with average biases at qPCR inputs representative of patient samples <9.7% across all assays while amplification efficiencies were within ±6% of the median. Assessments of reproducibility and precision were performed by testing 10 prostate cancer RNA samples over multiple instruments, reagent lots, operators, days (precision), and RNA input levels (reproducibility) using appropriately parameterized linear mixed models. The standard deviations for analytical precision and reproducibility were 1.86 and 2.11 GPS units (100-unit scale) respectively. CONCLUSIONS: The Oncotype DX Prostate Cancer Assay, a clinical RT-PCR assay specifically designed for use with prostate needle biopsies, has been analytically validated using very limited RNA inputs. The assay requirements and analytical performance will provide physicians with test results from a robust and reliable assay which will enable improved treatment decisions for men diagnosed with early-stage prostate cancer.

Biosurfactant-producing Bacillus are present in produced brines from Oklahoma oil reservoirs with a wide range of salinities.

Nine wells producing from six different reservoirs with salinities ranging from 2.1% to 15.9% were surveyed for presence of surface-active compounds and biosurfactant-producing microbes. Degenerate primers were designed to detect the presence of the surfactin/lichenysin (srfA3/licA3) gene involved in lipopeptide biosurfactant production in members of Bacillus subtilis/licheniformis group and the rhlR gene involved in regulation of rhamnolipid production in pseudomonads. Polymerase chain reaction amplification, cloning, and sequencing confirmed the presence of the srfA3/licA3 genes in brines collected from all nine wells. The presence of B. subtilis/licheniformis strains was confirmed by sequencing two other genes commonly used for taxonomic identification of bacteria, gyrA (gyrase A) and the 16S rRNA gene. Neither rhlR nor 16S rRNA gene related to pseudomonads was detected in any of the brines. Intrinsic levels of surface-active compounds in brines were low or not detected, but biosurfactant production could be stimulated by nutrient addition. Supplementation with a known biosurfactant-producing Bacillus strain together with nutrients increased biosurfactant production. The genetic potential to produce lipopeptide biosurfactants (e.g., srfA3/licA3 gene) is prevalent, and nutrient addition stimulated biosurfactant production in brines from diverse reservoirs, suggesting that a biostimulation approach for biosurfactant-mediated oil recovery may be technically feasible.