Isolation of enriched small RNA from cell-lysate using on-chip isotachophoresis

Ruba Khnouf; Crystal M Han; Sarah A Munro

doi:10.1002/elps.201900215

Isolation of enriched small RNA from cell-lysate using on-chip isotachophoresis

Electrophoresis. 2019 Dec;40(23-24):3140-3147. doi: 10.1002/elps.201900215. Epub 2019 Nov 14.

Authors

Ruba Khnouf^{1

2}, Crystal M Han^{3

4}, Sarah A Munro^{3

5}

Affiliations

¹ Department of Mechanical Engineering, Stanford University, Stanford, CA, United States.
² Department of Biomedical Engineering, Jordan University of Science and Technology, Irbid, Jordan.
³ Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, Stanford, CA, United States.
⁴ Department of Mechanical Engineering, San Jose State University, San Jose, CA, United States.
⁵ Minnesota Supercomputing Institute, University of Minnesota, MN, United States.

PMID: 31675123
DOI: 10.1002/elps.201900215

Abstract

In spite of the growing interest in the roles and applications of small RNAs (sRNAs), sRNA isolation methods are inconsistent, tedious, and dependent on the starting number of cells. In this work, we employ ITP to isolate sRNAs from the cell-lysate of K562 (chronic myelogenous leukemia) cells in a polydimethylsiloxane (PDMS) mesofluidic device. Our method specifically purifies sRNA of <60 nucleotides from lysate of a wide range of cell number spanning from 100 to 1 000 000 cells. We measured the amount of sRNA using the Agilent Bioanalyzer and further verified the extraction efficiency by reverse transcription quantitative PCR. Our method was shown to be more efficient in sRNA extraction than commercial sRNA isolation kits, especially when using smaller numbers of starting cells. Our assay presents a simple and rapid sRNA extraction method with 20 min assay time and no intermediate transfer steps.

Keywords: Isotachophoresis; Mesofluidic device; Small RNA; Small RNA extraction; Small RNA quantification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Humans
Isotachophoresis / methods*
K562 Cells
Microfluidic Analytical Techniques / methods*
RNA, Small Untranslated / analysis*
RNA, Small Untranslated / chemistry
RNA, Small Untranslated / isolation & purification*

Substances

RNA, Small Untranslated