Dr. Hirofumi Shintaku Profile

Dr. Hirofumi Shintaku

at Kyoto Univ

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Proceedings Article | 5 March 2015 Paper

Extraction and fractionation of RNA and DNA from single cells using selective lysing and isotachophoresis

Hirofumi Shintaku, Juan Santiago

Proceedings Volume 9320, 93200Q (2015) https://doi.org/10.1117/12.2084981

KEYWORDS: Luminescence, Microfluidics, Electrodes, Interfaces, Chemistry, Signal to noise ratio, Calibration, Process control, Data acquisition, Visualization

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Single cell analyses of RNA and DNA are crucial to understanding the heterogeneity of cell populations. The numbers of approaches to single cells analyses are expanding, but sequence specific measurements of nucleic acids have been mostly limited to studies of either DNA or RNA, and not both. This remains a challenge as RNA and DNA have very similar physical and biochemical properties, and cross-contamination with each other can introduce false positive results. We present an electrokinetic technique which creates the opportunity to fractionate and deliver cytoplasmic RNA and genomic DNA to independent downstream analyses. Our technique uses an on-chip system that enables selective lysing of cytoplasmic membrane, extraction of RNA (away from genomic DNA and nucleus), focusing, absolute quantification of cytoplasmic RNA mass. The absolute RNA mass quantification is performed using fluorescence observation without enzymatic amplification in < 5 min. The cell nucleus is left intact and the relative genomic DNA amount in the nucleus can be measured. We demonstrate the technique using single mouse B lymphocyte cells, for which we extracted an average of 14.1 pg total cytoplasmic RNA per cell. We also demonstrate correlation analysis between the absolute amount of cytoplasmic RNA and relative amount of genomic DNA, showing heterogeneity associated with cell cycle.

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