Diagnosis of malignancy in colon biopsy specimens by mRNA expression profiling

Background: The diagnosis of colon biopsy specimen is made by histology where the functional evaluation, genetic analysis of the samples can not be performed.

Aim: To develop an mRNA expression analysis by low cost arrays and array technology which can contribute to an enhanced diagnosis.

Materials and methods: Total RNA was extracted from healthy and diseased colonic biopsies from 10 patients with adenomatous polyps, 10 with colorectal adenocarcinoma and 10 with inflammatory bowel diseases (5 ulcerative colitis, 5 Crohn's disease). After quantity and quality control, the mRNA fraction was amplified by T7 RNA amplification method. RNA expression profile was evaluated by Atlas Glass microarrays (1081 genes). Principal component analysis and discriminant analysis was performed. Validation was done by real-time PCR.

Results: Significant overexpression of oncogenes (v-raf-1, c-met), growth factor genes (TGF-alpha, PDGF, VEGF, prostate differentiation factor), signal transduction molecule genes (NOS 2A, MAPKKK 5, ephrin-B2, GFR bound protein 2) and metabolism genes (glutation-S-transferase) was established during microarray analyses in colorectal cancer patients. Deleted in oral cancer tumor suppressor gene, genes involved in apoptosis (caspase-4 and caspase-8) and glutathione peroxidase was found significantly downregulated in colorectal cancer cases. Using discriminant analysis, based on the application of 5 components all of the cases could be classified correctly.

Conclusions: T7 RNA amplification is a reliable method to amplify mRNA from low cell number samples and the expressed genetic information does not alter under the protocol. LightCycler control is reliable to detect very low amounts of RNA. Atlas arrays with multivariate statistics can be used to diagnose mRNA expression profiles.