Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues: reproducible measurement of gene expression panels related to therapeutic responsiveness in clinical samples

C95 We evaluated and validated a novel platform that uses the branched-chain DNA amplification system for analysis of gene expression panels in clinical samples. We hypothesized that the clinical utility of an assay depends on its accuracy and reproducibility. The largest source for the development of effective diagnostic and biomarkers consists of formalin-fixed and paraffin embedded (FFPE) tissues. However, until recently, measurement of RNA in FFPE tissues has been unreliable. In contrast to other methods, the bDNA assay does not require RNA isolation from tissue homogenate, avoids measurement errors caused by enzymatic preamplification and has a simple workflow. To assess the reproducibility and sensitivity of the bDNA assay, samples from 10 prostate cancer xenografts and 4 FFPE tissue blocks were assayed by the bDNA assay and qPCR as a reference method. The validity and gene-specific sensitivity of the assay were further determined by analyzed a set prostate cancer specific genes in archival tissue blocks. To calculate the error of the bDNA assay, mixed effects statistical models were used to partition the overall variance into components attributable to xenograft, sample and assay. For FFPE tissues, the coefficient of reliability for 5 genes was significantly higher for the bDNA assay (93.0 - 100%) than for qPCR (82.4% - 95%0). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, and were similar to those from qPCRFROZEN and qPCRFFPE. The sensitivity of the bDNA assay in tissue homogenates was approximate 10-fold higher than in an equivalent amount of purified RNA. In macrodissected tissues from 9 - 13 year old blocks with poor RNA quality, the bDNA assay correctly identified the overexpression of known cancer genes. In conclusion, the bDNA assay is considerably more reliable and sensitive than qPCR and appears to be well suited for clinical analysis of FFPE tissues with diagnostic gene expression biomarker panels.