Examining Gene Expression Response to Therapeutic Compounds
The GeXP system consists of software for multiplex primer design, gene expression profiling, and visualization; reagents for setting up reverse transcription and multiplex PCR reactions; and hardware for the separation of amplified fluorescently-labeled DNA fragments by capillary electrophoresis.
The system uses a combined gene-specific and universal priming strategy that converts multiplexed PCR to a two-primer process using universal primers. As a result, the gene ratio in RNA samples is maintained during the PCR process. This strategy overcomes the variations in amplification efficiency of different genes during the conventional amplification process without compromising the detection sensitivity (Figure 1).
A rat toxicity panel was developed using the eXpress Designer software to target 22 genes involved in different pathway elements such as apoptosis, DNA damage response, stress response, drug detoxification, and cytotoxicity.
In addition, this plex includes three housekeeping genes (beta actin, GAPDH, and cyclophillin A) to be used as references in data analysis.
This gene expression system was used to study and compare all three thiazolidinediones that had been approved for type 2 diabetes treatment—Rezulin (troglitazone), Avandia (rosiglitazone), and Actos (pioglitazone). The purpose of the study was to examine specific gene expression responses to identify the potential different mechanisms responsible for the idiosyncratic toxicity of troglitazone versus pioglitazone and rosiglitazone.
Rat hepatocytes were used to observe the glitazone’s impact in metabolically active liver cells and the C9 cell line was utilized to see the effect of glitazones on a proliferating cell type. The C9 cell line is an epithelial cell line isolated from a normal liver of a young male rat. All cells were plated in a 96-well plate format and were treated at concentrations of 10, 50, and 100 µM for 24 hours.
After treatment, cells were harvested, and the total RNA from each well was extracted using the Agencourt® RNAdvance™ Cell Kit (Beckman Coulter). Expression levels of the selected genes determined by multiplex PCR reactions were compared with those of RNA from untreated control C9 and primary rat hepatocyte cells.