Kirk Molly J, Xu Chaoming, Paules Jonathan, Rothman Joel H
Department of Molecular Cellular and Developmental Biology, University of California, Santa Barbara;
Department of Molecular Cellular and Developmental Biology, University of California, Santa Barbara.
J Vis Exp. 2025 Jan 3(215). doi: 10.3791/66935.
The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes. To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on RNA-sequencing (RNA-seq), which can be performed on populations ranging from large cohorts to individual animals. Reverse transcription polymerase chain reaction (RT-PCR) and RNA interference (RNAi) are subsequently used to confirm RNA-seq findings and assess the genetic requirements for candidate genes, respectively. Such studies require an efficient, accurate, and affordable method for RNA extraction and measurement of relative transcript levels by quantitative RT-PCR (qRT-PCR). This work presents an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but also reduces the required time and cost for each extraction. This RNA extraction method yields quantities of cDNA that can be used to amplify and detect multiple transcripts by quantitative PCR (qRT-PCR). The method is validated by analyzing dynamic changes in the expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 β2 (HSP70 β2) and Heat-Shock Protein 90α (HSP90α), making it possible to assess their relative expression levels in heat-exposed individuals using qRT-PCR. STST effectively complements existing bulk and single tardigrade RNA extraction methods, permitting rapid and affordable examination of individual tardigrade transcriptional levels by qRT-PCR.
缓步动物模式种（Hypsibius exemplaris）是一种新兴的模式生物，以其在极端环境下的生存能力而闻名。为了探索这种极端耐受性的分子机制和遗传基础，许多研究依赖于RNA测序（RNA-seq），该技术可应用于从大群体到单个动物的样本。随后，逆转录聚合酶链反应（RT-PCR）和RNA干扰（RNAi）分别用于确认RNA-seq的结果，并评估候选基因的遗传需求。此类研究需要一种高效、准确且经济实惠的RNA提取方法，并通过定量RT-PCR（qRT-PCR）来测量相对转录水平。本文介绍了一种高效的单只缓步动物单管RNA提取方法（STST），该方法不仅能可靠地从单个缓步动物中分离出RNA，还能减少每次提取所需的时间和成本。这种RNA提取方法产生的cDNA量可用于通过定量PCR（qRT-PCR）扩增和检测多个转录本。通过分析编码两种热休克调节蛋白，即热休克蛋白70β2（HSP70β2）和热休克蛋白90α（HSP90α）的基因表达的动态变化，验证了该方法，从而能够使用qRT-PCR评估它们在热暴露个体中的相对表达水平。STST有效地补充了现有的批量和单只缓步动物RNA提取方法，允许通过qRT-PCR快速且经济地检测单个缓步动物的转录水平。
