Department of Mechanical Engineering, San Jose State University, San Jose, CA 95192, USA and Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, Stanford, CA, USA.
Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, Stanford, CA, USA.
Lab Chip. 2019 Aug 21;19(16):2741-2749. doi: 10.1039/c9lc00311h. Epub 2019 Jul 22.
We present an on-chip method for the extraction of RNA within a specific size range from low-abundance samples. We use isotachophoresis (ITP) with an ionic spacer and a sieving matrix to enable size-selection with a high yield of RNA in the target size range. The spacer zone separates two concentrated ITP peaks, the first containing unwanted single nucleotides and the second focusing RNA of the target size range (2-35 nt). Our ITP method excludes >90% of single nucleotides and >65% of longer RNAs (>35 nt). Compared to size selection using gel electrophoresis, ITP-based size-selection yields a 2.2-fold increase in the amount of extracted RNAs within the target size range. We also demonstrate compatibility of the ITP-based size-selection with downstream next generation sequencing. On-chip ITP-prepared samples reveal higher reproducibility of transcript-specific measurements compared to samples size-selected by gel electrophoresis. Our method offers an attractive alternative to conventional sample preparation for sequencing with shorter assay time, higher extraction efficiency and reproducibility. Potential applications of ITP-based size-selection include sequencing-based analyses of small RNAs from low-abundance samples such as rare cell types, samples from fluorescence activated cell sorting (FACS), or limited clinical samples.
我们提出了一种从低丰度样本中提取特定大小范围内 RNA 的片上方法。我们使用离子淌度电泳（ITP）和离子间隔物以及筛分基质来实现大小选择，从而以高收率获得目标大小范围内的 RNA。间隔区将两个浓缩的 ITP 峰分开，第一个峰包含不需要的单核苷酸，第二个峰聚焦于目标大小范围（2-35nt）的 RNA。我们的 ITP 方法可排除 >90%的单核苷酸和 >65%的较长 RNA（>35nt）。与使用凝胶电泳进行的大小选择相比，基于 ITP 的大小选择可使目标大小范围内提取的 RNA 量增加 2.2 倍。我们还证明了基于 ITP 的大小选择与下游下一代测序的兼容性。与通过凝胶电泳进行大小选择的样品相比，基于芯片 ITP 制备的样品在转录物特异性测量的重现性方面更高。我们的方法为测序提供了一种有吸引力的替代传统样品制备方法，其具有更短的测定时间、更高的提取效率和重现性。基于 ITP 的大小选择的潜在应用包括对来自低丰度样本（如稀有细胞类型、荧光激活细胞分选（FACS）样本或有限的临床样本）的小 RNA 进行测序分析。
