Use of a rainbow trout oligonucleotide microarray to determine transcriptional patterns in aflatoxin B1-induced hepatocellular carcinoma compared to adjacent liver.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its occurrence is associated with a number of environmental factors including ingestion of the dietary contaminant aflatoxin B(1) (AFB(1)). Research over the last 40 years has revealed rainbow trout (Oncorhynchus mykiss) to be an excellent research model for study of AFB(1)-induced hepatocarcinogenesis; however, little is known about changes at the molecular level in trout tumors. We have developed a rainbow trout oligonucleotide array containing 1672 elements representing over 1400 genes of known or probable relevance to toxicology, comparative immunology, carcinogenesis, endocrinology, and stress physiology. In this study, we applied microarray technology to examine gene expression of AFB(1)-induced HCC in the rainbow trout tumor model. Carcinogenesis was initiated in trout embryos with 50 ppb AFB(1), and after 13 months control livers, tumors, and tumor-adjacent liver tissues were isolated from juvenile fish. Global gene expression was determined in histologically confirmed HCCs compared to noncancerous adjacent tissue and sham-initiated control liver. We observed distinct gene regulation patterns in HCC compared to noncancerous tissue including upregulation of genes important for cell cycle control, transcription, cytoskeletal formation, and the acute phase response and downregulation of genes involved in drug metabolism, lipid metabolism, and retinol metabolism. Interestingly, the expression profiles observed in trout HCC are similar to the transcriptional signatures found in human and rodent HCC, further supporting the validity of the model. Overall, these findings contribute to a better understanding of the mechanism of AFB(1)-induced hepatocarcinogenesis in trout and identify conserved genes important for carcinogenesis in species separated evolutionarily by more than 400 million years.

Strategies for genotyping: Effectiveness of tailing primers to increase accuracy in short tandem repeat determinations.

A problem associated with automated analysis of fluorescently labeled fragments separated by slab gel or capillary electrophoresis is the doublet peak formed when Taq DNA Polymerase adds a nontemplated nucleotide (generally an adenosine) to the 3' end of the product.This nontemplated addition (plus A) is primarily dependent on the 5' sequence of the reverse primer and, to a lesser extent, polymerase chain reaction (PCR) conditions. Primers may amplify the true product, the plus A product or a doublet product comprised of both. When using markers based on dinucleotide repeats, this single base pair difference can make binning and accurate automated analysis problematic. To drive the PCR reaction consistently to the plus A product, the sequence of the nonfluorescent primer used in amplification can be modified by adding a 5' tail favoring the nontemplated addition. The present study, conducted by the Fragment Analysis Research Group (FARG) of the Association of Biomolecular Resource Facilities, provided researchers with an opportunity to compare normal products amplified with a dinucleotide marker to products amplified with the same primer to which a 5' tail designed to promote the plus A product had been added. The study also included a sample amplified with a tetranucleotide repeat marker for comparison. The results from this study were returned to the FARG for comprehensive analysis and are reported here.