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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 &gt;90% of single nucleotides and &gt;65% of longer RNAs (&gt;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.

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