Improved Sensitivity for Staining RNA with SYBR Gold Stain ( RNA gel system FMC). The gel was run...)

Improved Sensitivity for Staining RNA with SYBR Gold Stain

RNA gel system, FMC). The gel was run at 100 V for one hour in 1X MOPS, then stained for one hour with SYBRgold nucleic acid gel stain. The stain solution (50 l of SYBR Gold stock, 450 l of 5 M NaOH, and 250 l of 1X MOPS) can be stored in the dark for up to 48 hours.

To compare SYBR Gold stain to ethidium bromide staining, two identical gels were run with various amounts of total RNA: One gel was stained with SYBR Gold (described above), and the second gel was stained with 5 ng/ml of ethidium bromide in distilled water for 20 minutes at room temperature, then destained in water for 20 minutes. The stained gels were viewed on an Eagle Eye II still video system using the SYBR Green band-pass filter or the ethidium bromide band-pass filter.

The live gel image was initially captured under fluorescent light using the REAL TIME IMAGING feature of the Eagle Eye II. After determining the correct gel orientation, and adjusting the zoom, focus, and approximate exposure, the gel was exposed to UV light. The INTEGRATION function was then used to acquire the final image by setting the integration to 10 frames at 1/30 second. While the image was integrating, the camera exposure was adjusted until saturation was reached [saturation is denoted by red regions on the imaging screen]. After integration was terminated, the image was saved and printed.

SYBR Gold vs. Ethidium Bromide

In Figure 1, an image of a SYBR Gold stained RNA gel on the Eagle Eye II clearly shows 250 ng of poly(A)⁺ RNA from a number of samples. To assess the quality of the poly(A)⁺ RNA, we prefer to run 250 ng of RNA which, for a variety of samples, routinely produces a clear image well above the limit of sensitivity. To assess the relative sensitivity of the SYBR Gold stain, various amounts of total RNA were
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( RNA gel system FMC). The gel was run...)Improved Sensitivity for Staining RNA with SYBR Gold Stain

SYBR Gold vs. Ethidium Bromide