Gene expression profiling analysis is becoming one of the most frequently used tools for functional genomics. However, this kind of analysis is most commonly performed one gene at a time.
Beckman Coulter (www.beckmancoulter.com)and Althea Technologies (www.altheatech.com) developed a technique and a system that enable multiplexed PCR for the expression analysis of 20–30 genes in a single reaction in a 96-well plate. The GenomeLab™ GeXP Genetic Analysis system delivers accurate and quantitative results for hundreds or even thousands of samples using very small amounts of total RNA.
The GenomeLab eXpress Profiling software, with proprietary algorithms, enables customized primer design and multiplex assembly. Using this program, we developed a rat toxicity panel containing 22 genes involved in multiple toxicity related pathways. The new kit, the GenomeLab GeXP Rat MultitoxPlex, contains 23 genes plus three reference genes and one internal control.
As an example, we will describe here a study that examined specific gene expression responses to troglitazone, an FDA-approved type 2 diabetes drug that was removed from the market in 2000 after significant adverse effects were observed in some patients.
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.
Description of Method
Total RNA samples of 25 nanograms from compound-treated and untreated primary rat hepatocytes were amplified using GeXP Rat MultitoxPlex reagents. Amplified, dye-labeled fragments were separated by capillary electrophoresis. Gene-specific peaks were identified, quantitated, and normalized, and gene expression profiles were created and visualized using the system’s software.
There are five basic steps involved:
• Primer design. Primers for the multiplexed panel are designed by importing the target gene accession numbers or gene IDs into the eXpress Designer module of the eXpress Profiler software. The primers are designed to have similar GC content and melting temperature and to generate amplified products five to seven base pairs apart, and in the range of 100–400 nt.
• cDNA synthesis. Following total RNA extraction, gene-specific cDNA synthesis is performed using the custom reverse-transcriptase (RT) reaction mix containing 26 chimeric reverse primers. Each chimeric primer has a 19 nt universal tag sequence at its 5´ end and gene-specific sequence at its 3´ end. The RT reaction (multiplex cDNA synthesis) is performed in a thermal cycler using the following incubation program: 48°C for 1 min ’ 37°C for 5 min ’ 42°C for 60 min ’ 95°C for 5 min ’ hold at 4°C.
• PCR. An aliquot (9.3 µL) of the RT reaction product is then transferred to the PCR reaction mix (10.7 µL). The final PCR reaction mixture contains 26 pairs of chimeric reverse and forward primers as well as one pair of universal primers (D4-labeled universal forward primer and un-labeled universal reverse primer). Fluorescently labeled amplicons were generated in PCR reactions using the following thermal cycling program: 95°C for 10 min ’ (94°C for 30 sec ’ 55°C for 30 sec ’ 68°C for 1 min) x 35 cycles ’ hold at 4°C.
• Separation by the GenomeLab GeXP Genetic Analysis System. Fluorescently labeled PCR products are diluted 1:40 in a mixture containing size standard 400 and sample loading solution (SLS) and separated via capillary electrophoresis.
• Fragment Analysis and eXpress Profiling Analysis. Once separated, the data is initially analyzed using the fragment analysis software. Peak area information is then imported to eXpress Analysis in which gene IDs are associated with peak area information. After normalization to one of the three housekeeping genes, the results can be exported or viewed using the eXpress Map module of the eXpress Profiler software where the data can be visualized in several ways.
Discussion of Results
In this study we sought to determine if data obtained from monitoring the impact of troglitazone in treated cells in culture with gene expression analysis would suggest mechanisms of in vivo drug toxicity. This study included performing the same analysis with the currently available glitazones, pioglitazone, and rosiglitazone, to compare potential toxicity profiles of the three drugs.
Figure 2 shows the gene expression profile obtained in Glitazone-treated Rat C9 cells. The results show a marked upregulation of Nqo1, Gadd45, Gadd153, p21, COX-2 and Ho1 by Troglitazone, not by rosiglitazone or pioglitazone in C9 cells. There was also limited inductionof CYP2B1 and aldehyde DH, genes involved in drug metabolism, by troglitazone as compared to pioglitazone and rosiglitazone.
Figure 3 shows the gene expression profile obtained in glitazone-treated rat primary hepatocytes. Similar to results obtained in C9 cells, significant upregulation of Nqo1, Gadd45, Gadd153, Ho1 and p21 by troglitazone, not by rosiglitazone or pioglitazone, was detected in primary rat hepatocytes. In addition, a slight downregulation of CyclinD1 was also observed.
Our results suggest that the impact of troglitazone on the expression of the genes monitored reveals a profile of potential toxicity that is not apparent when analyzing the gene profiles produced by rosiglitazone and pioglitazone in vitro. This observation is consistent with the toxicity of troglitazone, but not rosiglitazone and pioglitazone, suggesting that the Rat MultitoxPlex was able to accurately capture compound-induced gene expression changes that were predictive of potential toxicity in vivo.
The gene expression screen that was performed in vitro with troglitazone previously predicted that the drug could be toxic by a variety of mechanisms. One of the current explanations of idiosyncratic toxicity is the “multiple determinant hypothesis.” This hypothesis predicts the probability that multiple independent biochemical events could synergistically contribute to the mechanisms by which troglitazone becomes toxic in a small percentage of the human population. The gene expression data presented here provides support for the multiple determinant hypothesis with different gene responses indicating a plurality of mechanisms of troglitazone toxicity in small population subsets.
The study demonstrates that the GenomeLab Rat MultitoxPlex panel can be an effective tool in predictive toxicity studies of different pathways or mechanisms within a model system.
The PCR process is covered by patents owned by Roche Molecular Systems and F. Hoffman-LaRoche.