Simultaneous RNA purification and size selection using on-chip isotachophoresis with an ionic spacer.

Autor:	Han CM; 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. msalit@stanford.edu., Catoe D; Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, Stanford, CA, USA. msalit@stanford.edu., Munro SA; Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, Stanford, CA, USA. msalit@stanford.edu and Minnesota Supercomputing Institute, University of Minnesota, MN 55455, USA., Khnouf R; Department of Biomedical Engineering, Jordan University of Science and Technology, Irbid, Jordan and Department of Mechanical Engineering, Stanford University, Stanford, CA 94305, USA., Snyder MP; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA., Santiago JG; Department of Mechanical Engineering, Stanford University, Stanford, CA 94305, USA., Salit ML; Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, Stanford, CA, USA. msalit@stanford.edu., Cenik C; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA and Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78705, USA. ccenik@austin.utexas.edu.
Jazyk:	angličtina
Zdroj:	Lab on a chip [Lab Chip] 2019 Aug 21; Vol. 19 (16), pp. 2741-2749. Date of Electronic Publication: 2019 Jul 22.
DOI:	10.1039/c9lc00311h
Abstrakt:	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.
Databáze:	MEDLINE
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