A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
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DOI：
10.1089/nat.2015.0578
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发表时间：
2016-04-01
影响因子：
4
通讯作者：
Khvorova, Anastasia
中科院分区：
文献类型：
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作者：
Coles, Andrew H.;Osborn, Maire F.;Khvorova, Anastasia
基于RNA干扰(RNAi)的治疗方法的临床前发展需要一种快速、准确和可靠的方法来同时定量数百个样本中的mRNA敲除。实现这一点的最成熟的方法是定量实时聚合酶链式反应(qRT-PCR)，这是一种需要样品纯化的劳动密集型方法，这增加了引入额外偏差的可能性。在这里，我们描述了与96孔Qiagen TIssueLyser II相连的Quantigene((R))分支DNA(BDNA)分析是一种快速且可重复性的替代qRT-PCR的方法，用于直接从组织活检组织中定量分析体内的mRNA表达。BDNA分析是一种高通量、基于平板的发光技术，能够直接测量来自各种生物样本的组织裂解产物的mRNA水平。我们已经对这项技术进行了系统的评估，用于体内检测基于RNAi的沉默。我们表明，从纯化的RNA和组织裂解产物中也可以获得类似的质量数据。一般来说，我们观察到动物内和动物间的变异性较低(对照样品约为10%)，而中等精密度较高。这使得评估体内寡核苷酸有效性的样本量降到最小。
Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene((R)) branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo.