In-situ sampling and separation of RNA from individual mammalian cells.
In this investigation RNA was directly sampled and separated at the single-cell level (without extraction) by capillary electrophoresis (CE). Laser-induced fluorescence (LIF) was employed to detect ethidium bromide-labeled RNA molecules under native conditions. Hydroxypropylmethylcellulose was used as a matrix for molecular sieving. Additives to the polymer solution included poly(vinylpyrrolidone) to eliminate the electroosmotic flow and mannitol to enhance the separation. Peak identities were confirmed as RNA by enzymatic treatment with RNase I. The individual Chinese Hamster Ovary (CHO-K1) cells were injected into a capillary and the cells were lysed online with sodium dodecyl sulfate (SDS) solutions before running electrophoresis. Low molecular mass (LMM) RNAs as well as larger fragments (tentatively identified as 18S and 28S ribosomal RNA by comparison with the literature) were detected with this system, which corresponds to a detected amount of approximately equals 10-20 pg of RNA/cell. A Proteinase K study showed that proteins incorporated with RNA molecules were eliminated by SDS treatment and thus did not influence the migration of RNA. Experiments were also performed with this technique to detect nucleic acid damage. Changes in the peak pattern were detected in the cells treated with hydrogen peroxide, which meant that strand breaks occurred in DNA and RNA. It was found that 60 mM caused the most severe damage to the nucleic acids.
Han
F
F
Department of Chemistry, University of California, Riverside 92521, USA.
Lillard
S J
SJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004249:DNA Damage; D019075:Electrophoresis, Capillary; D005453:Fluorescence; D007834:Lasers; D012313:RNA / Q000302:isolation & purification*
D012313:RNA