High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology.
High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology.
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DOI：
10.3791/61132
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发表时间：
2020-05-28
期刊：
影响因子：
--
通讯作者：
Casanueva O
中科院分区：
文献类型：
--
作者：
Chauve L;Le Pen J;Hodge F;Todtenhaupt P;Biggins L;Miska EA;Andrews S;Casanueva O
本文提出了一种快速、稳健且高度灵敏的秀丽隐杆线虫高通量逆转录定量PCR（RT-qPCR）检测方法。该方案获得精确的测量基因表达从单个蠕虫或从批量样品。这里提出的协议提供了一种新的适应现有的方法互补DNA（cDNA）的制备耦合到纳米流体RT-qPCR平台。该方案的第一部分，命名为“蠕虫-CT”，允许直接从线虫产生cDNA，而不需要预先分离mRNA。它通过允许在3.5小时内从96个蠕虫制备cDNA来增加实验通量。该方案的第二部分使用现有的纳米流体技术对cDNA进行高通量RT-qPCR。本文评估了两种不同的纳米流体芯片：第一次运行96个样本和96个目标，在大约1.5天的实验室工作中产生了9，216个反应。第二种芯片类型由六个12 x 12阵列组成，产生864个反应。在这里，蠕虫到CT的方法是通过量化编码热休克蛋白的基因的mRNA水平从单个蠕虫和批量样品证明。提供了一个广泛的引物列表，其设计用于扩增C.线虫基因组
This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named ‘Worm-to-CT’, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.
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