Chapter 4 - Measuring Effects at the Gene Transcription Level

Abstract

The evaluation of changes in gene expression at the transcriptome level (mRNA) has become an active field in both the laboratory and field investigations in ecotoxicology. Ecotoxicogenomics represent the field of ecotoxicology interested in the study of changes in gene expression levels of a high number of genes in the attempt to understand the mode of action of xenobiotics at the fundamental level. High-throughput gene transcription analysis is achieved using microarrays, which can measure more than 10,000 genes transcripts at a time. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) represents a very sensitive and more specific means for gene transcription analysis but for a more limited number of transcripts (usually <100 gene targets at a time). The analysis of gene expression by qRT-PCR is more amenable to laboratory (less expensive) since the instrument is becoming less expensive to acquire. However, these methods require a priori knowledge of gene sequence to prepare specific oligonucleotides primers to initiate the reaction. If the target sequences are not known the use of complementary sequences from other species or the use of degenerate primers can be used. Guidelines are presented to extract RNA devoid of DNA from various tissues and the general principles to determine the concentration of mRNA in tissues using the qRT-PCR approach. A troubleshooting guide is also included for all steps involved in gene expression analysis such as primer design, RNA extraction/purity/integrity, and recommended temperatures during amplification steps.