Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser-induced fluorescence.
In this work, five fluorescent dyes (SYTO-9, SYBR Green I, SYBR Green II, SYBR Safe, and SYBR Gold) were used as both on-column and precolumn stains for total RNA analysis by CE-LIF with Ar ion laser excitation. In the on-column RNA stain, the SYTO-9 provided the highest fluorescence intensity and the lowest detectable concentration, as low as 10 pg/μL, while the SYBR Green II and SYBR Gold were adsorbed on the poly(ethylene oxide) thus affected the separation efficiency. As a precolumn stain, SYBR Gold was the most sensitive among the five dyes due to the strong affinity between the dye and RNA molecules. As a result, a single-cell quantity of RNA (10-30 pg per cell) could be detected by CE-LIF with precolumn staining by SYBR Gold. Because of the great savings of fluorescent dye using precolumn stain (one button dye may use for one million stain), this method is the best strategy for RNA staining in terms of cost-effectiveness and sensitivity.
Department of Chemistry, Tunghai University, Taichung, Taiwan.
Yi-An
YA
Chung
Department of Chemistry, Tunghai University, Taichung, Taiwan.
Yi-Hsin
YH
Chen
Department of Chemistry, Tunghai University, Taichung, Taiwan.
Po-Ling
PL
Chang
D001128:Argon / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D011092:Polyethylene Glycols; D012313:RNA / Q000032:analysis*
D005456:Fluorescent Dyes; D011092:Polyethylene Glycols; D012313:RNA; D001128:Argon
Capillary electrophoresis; Fluorescent dye; Laser-induced fluorescence; Poly(ethylene oxide); Ribonucleic acid