On-chip separation and analysis of RNA and DNA from single cells.
The simultaneous analysis of RNA and DNA of single cells remains a challenge as these species have very similar physical and biochemical properties and can cross-contaminate each other. Presented is an on-chip system that enables selective lysing of single living cells, extraction, focusing, and absolute quantification of cytoplasmic RNA mass and its physical separation from DNA in the nucleus using electrical lysing and isotachophoresis (ITP). This absolute quantitation is performed without enzymatic amplification in less than 5 min. The nucleus is preserved, and its DNA fluorescence signal can be measured independently. We demonstrate the technique using single mouse lymphocyte cells, for which we extracted an average of 14.1 pg of total RNA per cell. We also demonstrate correlation analysis between the absolute amount of RNA and relative amount of DNA, showing heterogeneity associated with cell cycles. The technique is compatible with fractionation of DNA and RNA and with downstream assays of each.
Department of Mechanical Engineering, ‡Divisions of Blood and Marrow Transplantation, and §Department of Chemical Engineering, Stanford University , Stanford, California 94305, United States.
Hirofumi
H
Shintaku
Hidekazu
H
Nishikii
Lewis A
LA
Marshall
Hidetoshi
H
Kotera
Juan G
JG
Santiago
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D008214:Lymphocytes / Q000737:chemistry*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis*
D012313:RNA; D004247:DNA