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Multiplexed RT- PCR for High Throughput Screening Applications

机译：高通量筛选应用的多重RT-PCR

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摘要

To increase efficiency of high throughput gene expression profiling, we established a new TaqMan RT-PCR (real-time reverse transcriptase-polymerase chain reaction with internal probes for the quantification of PCR products) method for quantitative mRNA expression analysis. In this procedure, we utilized poly-A mRNA capture plates and validated a multiplexed single tube RT-PCR assay for cell culture applications, including compound testing via gene induction measurement. In the described procedure, all steps including RNA extraction, RT and PCR are performed in the same tube, thus significantly enhancing throughput of this method. Optimization of conditions, including the number of cells necessary for detection of mRNA signal was performed. With a relatively abundant message such as GAPDH, we saw a linear response for all of the concentrations tested, from 10,000 cells to 10 cells. We have also demonstrated multiplexing of different targets within the PCR reactions. In these experiments, we combined VIC-labeled probes for GAPDH with several FAM-labeled probes obtained from Assays On Demand (Applied Biosystems). In the reported experiments, multiplexing did not affect the efficiency of RT-PCR. We also demonstrated the utility of this technology for compound screening applications. The described technology also has the potential to accelerate studies on target and biomarker identification and toxicity assessment in ADMET (absorption, distribution, metabolism, elimination, and toxicity) testing.
机译：为了提高高通量基因表达谱分析的效率，我们建立了新的TaqMan RT-PCR（实时逆转录聚合酶链反应，内部探针用于定量PCR产物的定量）方法，用于定量mRNA表达分析。在此程序中，我们利用了poly-A mRNA捕获板，并验证了用于细胞培养应用的多重单管RT-PCR分析，包括通过基因诱导测量的化合物测试。在所描述的过程中，包括RNA提取，RT和PCR在内的所有步骤均在同一管中执行，因此显着提高了该方法的通量。优化条件，包括检测mRNA信号所需的细胞数量。有了相对丰富的信息（例如GAPDH），我们看到了从10,000个细胞到10个细胞的所有浓度测试的线性响应。我们还证明了PCR反应中不同靶标的多重化。在这些实验中，我们将GAPDH的VIC标记探针与从“按需检测”（Applied Biosystems）获得的几种FAM标记探针结合在一起。在报道的实验中，多路复用并不影响RT-PCR的效率。我们还展示了该技术在化合物筛选应用中的实用性。所描述的技术还具有加速ADMET测试（吸收，分布，代谢，消除和毒性）中靶标和生物标志物鉴定以及毒性评估的研究的潜力。

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