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| Integrity of Gene Expression Patterns from Small Samples of Distinct Tumor Types with Nugen OVATION RNA Amplification System (ORAS) |
| Topic:
PCR methods |
X. Wang1, B. McCarthy1, S. Paul1, N. Chiorazzi1, A. Rawstron2, L. Goodwin1;
1North Shore-LIJ Research Institute, Manhasset, NY, United States, 2Hematological Malignancy Diagnostic Service, Leeds, United Kingdom. |
| Poster Board Number: P104-M |
Background: Clinical samples present a unique opportunity to establish gene expression patterns in specific cancers with predictive clinical outcomes. Often these samples are precious, with low RNA yield (<200ng/needle aspirate), less than required to analyze a single target gene’s expression by Quantitative PCR (QPCR). ORAS enriches linearly the amount of mRNA species while maintaining relative gene expression levels compared to the unamplified material. We assessed the integrity of gene expression patterns by QPCR analysis from a subset of genes altered in specific cancers from both amplified and unamplified RNA samples.
Material and Methods: ~ 5ul total RNA (20-100ng) from 11 samples of family members of patients with specific cancers, B cell lymphoma, breast cancer and 3 normal controls were available for testing ORAS generation of cDNA. Gene expression levels of twelve genes associated with cell cycle, apoptosis and signaling were measured using QPCR, with b-actin as reference.
Results: ORAS generated enough cDNA from small sample RNA for 20-30 QPCR analyses. 12 genes (low, medium and abundant) expression patterns from amplified RNA were compared with total RNA from two cancer cell lines. Linear correlation of differential gene expression between the two was R2 0.925. There was a reproducible pattern of five genes with altered expression, characteristic of specific cancer type, found in family member samples but not in the other cancers tested. This gene expression panel may provide a biomarker for diagnosis of clinically undetectable disease and prognosis of outcome for family members of patients with specific cancers. |
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