Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser‐induced fluorescence

Abstract

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.

Determination of live and dead Komagataeibacter xylinus cells and first attempt at precise control of inoculation in nanocellulose production

“…SG is a commonly used fluorochrome for staining nucleic acids in agarose gels within molecular biology techniques. SG has been reported to bind dsDNA more tightly and efficiently than SYTO 9 (Monis et al , ) and to have a stronger fluorescence intensity of DNA/dyes compared with SYBR Gold, SYBR Green II, SYBR Safe and SYTO 9 under identical excitation (Barbesti et al , ; Chung et al , ). The purpose of using the SYBR Green I/PI assay is to assess whether the combination works similarly to the SYTO 9/PI (LIVE/DEAD ® BacLight TM commercial kit) assay and has a comparable or even better efficiency when used on the K. xylinus strains.…”