Advantages of RT-PCR
Oncotype DX® analyzes the expression of a panel of 21 genes from a tumor specimen using a technique called RT-PCR (reverse transcriptase-polymerase chain reaction). A high-throughput, real-time RT-PCR method was developed to analyze the expression of several hundred genes simultaneously. It is sensitive, specific, highly reproducible, and has a wide dynamic range. RT-PCR is a mature technology that is routinely utilized in several clinical applications including viral load testing for HIV.
To quantify gene expression, RNA is extracted from formalin-fixed, paraffin-embedded (FPET) tumor tissue and subjected to DNase I treatment. Total RNA content is measured and the absence of DNA contamination is verified. Reverse transcription is performed and is followed by quantitative TaqMan® RT-PCR reactions in 384-well plates. The expression of each of 16 genes is measured in triplicate and then normalized relative to a set of five reference genes.
The Oncotype DX standardized testing methods have been optimized to minimize variablilty due to:
- Tissue preparation method: FPET vs. fresh frozen
- Tumor block age, storage, and variability in preparation
- Heterogeneity within and between FPET blocks
- Heterogeneity with respect to enriched tumor and non-tumor areas within an FPET block
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