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Elena Barbaria, Grazia Saturno, Daniela Radice Introduction The failure of promising drug candidates, mostly related to unpredicted toxicity, is extremely expensive for pharmaceutical companies. Therefore, detailed data about the changes in gene expression of toxicological relevant marker genes today take a substantial place in toxicological and pharmacological evaluation. Almost all pharmaceutical companies are developing strategies for early toxicological screening; features, such as apoptosis, oxidative stress, DNA damage or drug-drug interactions are considered during the process of compound selection. Our strategy is aimed to describe an expression pattern of 500 relevant marker genes in 50 different tissues derived from pre-clinical species. We use Real-Time Polymerase Chain reaction (PCR*) as a screening technique. This method allows quantitation of gene expression during amplification of the cDNA, and is more precise, sensitive and specific than comparable techniques. Furthermore, it is easy to automate for high-throughput screening. Materials and Methods Total RNA was isolated from tissues with RNeasy Mini Kit (Qiagen), and treated with DNase (Qiagen) to avoid genomic DNA contamination. RNA integrity was checked with a Bioanalyzer 2001 (Agilent Technologies), using RNA 6000 Nano Kit. RNA yield was determined with RiboGreen RNA Quantitation Kit (Molecular Probes). Retrotranscription was performed using SuperScript II RNaseH- Reverse Transcriptase (Invitrogen) following manufacturer’s instructions.
Every cDNA diluted 1:100 was used as template in a TaqMan PCR reaction (prepared with TaqMan Universal PCR Master Mix, Applied Biosystems) using specific primers for different genes. Standard curves were obtained with serial dilutions of a pool of cDNA samples derived from each tissue, to quantify the relative expression of target genes. Assays were carried out using ABI Prism 7900 sequence detection system (Applied Biosystems). RNA amount of each target gene was normalized versus the expression of Ribosomal RNA 18S (Ribosomal RNA Control Reagents, Applied Biosystems). Results and Discussion Protocol Setup. The requirement to process a large amount of samples led us to evaluate two different liquid handling systems in order to implement both time and cost effectiveness, and system reliability. We compared experiments performed on a Biomek 2000 and a competitor's liquid handler. The protocol consisted of a PCR assay with primers and probe specific for a housekeeping gene (Ribosomal RNA 18S) on replicates of the same cDNA sample. Preliminary data showed reproducibility with a 30% lower error in assays prepared with Biomek 2000 than in those prepared with the second automation system (data not shown). Thus, subsequent tests were done on the Biomek 2000.
TaqMan PCR Universal Master Mix was manually prepared and dispensed into a 96-well plate. Then, using the P200L tool, we created a brief method to automatically dispense 200 µL of cDNA from 1.5 mL tubes to a second 96-well plate. The MP20 tool was used to prepare PCR reactions, mixing 5 µL of cDNA and 7.5 µL of Master Mix in each well of a 384-well plate. As negative control, we prepared additional reactions without cDNA template. Due to the low volumes and the viscosity of the Master Mix we observed some variability in the actual dispensed volume. To address this problem, we adjusted the calibration values of the MP20 decreasing the slope of the calibration curve of the tool from 6.67 to 5.97, and we reduced the dispensing rate in both source and destination from 7 to 3. These changes resulted in a drop of the dead volume from 30% to 15% of the total Mix volume. High-Throughput Real-Time PCR Setup Following these initial tests we developed a fully automated method to dispense cDNA and Master Mix. This allowed us to prepare 10 384-well plates in one batch in order to carry out high-throughput Real-Time PCR analysis on 10 different target genes. Preparation of every PCR Master Mix (containing primers and probe specific for different genes) and distribution in each column of two 96-well plates were carried out manually, while cDNA samples were automatically dispensed from 1.5 mL tubes to each well of two 96-well plates. The initial deck configuration of the method is shown in Figure 2. PCR reactions were prepared as previously mixing 5 µL of each cDNA and 7.5 µL of each Master Mix in all 384-well plates. Three 384-well plates are prepared within two hours and immediately sealed by the integrated plate sealer and stored in the stacker carousel. Within seven hours 10 plates, each containing a Mix specific for a different target gene, are prepared and ready to be analyzed by the ABI Prism 7900.
Assays developed with this method show high reproducibility and excellent efficiency. Figure 3 shows a standard curve of Ribosomal RNA 18S assay performed on triplets of five sequential dilutions of the same cDNA. The linear correlation is 0.9995. The amplification plot of the same assay is shown in figure 4, corresponding Ct values in table 1.
Acknowledgement We would like to thank Emanuele Oggioni from Beckman Coulter Italy for excellent technical support. * All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffmann-LaRoche, Ltd. For comments or questions about T3 Update, please contact David Daniels, Ph.D., editor.
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