Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening
Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening
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DOI：
10.1177/1087057112465184
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发表时间：
2013-04-01
影响因子：
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通讯作者：
van Beusechem, Victor W.
中科院分区：
文献类型：
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作者：
Siebring-van Olst, Ellen;Vermeulen, Christie;van Beusechem, Victor W.
萤火虫荧光素酶基因通常用于基于细胞的报告基因检测。用于高通量筛选 (HTS) 的方便的荧光素酶测定试剂已市售。然而，这些试剂的高昂成本并不是一些学术实验室能够承受的。因此，我们着手开发一种经济实惠的荧光素酶检测试剂，适用于 HTS 格式，使用简单的液体处理步骤。该试剂由单独的化学成分自制，并针对发光强度和稳定性进行了优化。我们确定了最昂贵成分二硫苏糖醇 (DTT) 和 D-荧光素的最低浓度，从而使每个样品的总检测试剂成本低于 1 美分。通过省略辅酶 A 和降低 DTT 浓度，可以最大限度地提高信号稳定性。使用携带 p53 依赖性荧光素酶报告基因构建体和调节 p53 转录活性的 siRNA 的两种癌细胞系在高通量环境中验证了该测定法。通过沉默 PPM1D 或 SYVN1 诱导 p53 活性以及通过沉默 p53 降低 p53 活性在 2 小时测量期内保持恒定，具有良好的测定质量（Z' 因子大多高于 0.5）。因此，本文描述的荧光素酶测定可用于基于细胞的 HTS 中经济实惠的报告读数。
The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z' factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.