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Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate)

Author:
Subramanian Parimalam, Sangamithirai, Oguchi, Yusuke, Abdelmoez, Mahmoud N., Tsuchida, Arata, Ozaki, Yuka, Yokokawa, Ryuji, Kotera, Hidetoshi, Shintaku, Hirofumi
Source:
Analytical chemistry 2018 v.90 no.21 pp. 12512-12518
ISSN:
1520-6882
Subject:
RNA, dithiothreitol, electric field, hydrodynamics, neoplasm cells, plasma membrane, propionic acid, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, reverse transcription
Abstract:
We present a microfluidic method for electrical lysis and RNA extraction from single fixed cells leveraging reversible cross-linker dithiobis(succinimidyl propionate) (DSP). Our microfluidic system captures a single DSP-fixed cell at a hydrodynamic trap, reverse-cross-links the DSP molecules on a chip with dithiothreitol, lyses the plasma membrane via electrical field, and extracts cytoplasmic RNA with isotachophoresis-aided nucleic acids extraction. All of the on-chip processes complete in less than 5 min. We demonstrated the method using K562 leukemia cells and benchmarked the performance of RNA extraction with reverse transcription quantitative polymerase chain reaction. We also demonstrated the integration of our method with single-cell RNA sequencing.
Agid:
6201546