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-in duced fluorescence (LIF) was employed to detect ethidium bromide-labeled RN A molecules under native conditions. Hydroxypropylmethylcellulose was used as a matrix for molecular sieving. Additives to the polymer solution includ ed poly(vinylpyrrolidone) to eliminate the elctroosmotic now and mannitol t o enhance the separation. Peak identities were confirmed as RNA by enzymati c treatment with RNase I. The individual Chinese Hamster Ovary (CHO-K1) cel ls were injected into a capillary and the cells were lysed online with sodi um dodecyl sulfate (SDS) solutions before running electrophoresis. Low mole cular mass (LMM) RNAs as well as larger fragments (tentatively identified a s 18S and 28S ribosomal RNA by comparison with the literature) were detecte d with this system, which corresponds to a detected amount of approximate t o 10-20 pg of RNA/cell. A Proteinase K study showed that proteins incorpora ted with RNA molecules were eliminated by SDS treatment and thus did not in fluence the migration of RNA. Experiments were also performed with this tec hnique to detect nucleic acid damage. Changes in the peak pattern were dete cted in the cells treated with hydrogen peroxide, which meant that strand b reaks occurred in DNA and RNA. It was found that 60 mM caused the most seve re damage to the nucleic acids.