Eagle Eye II still video system provides quality images from nanogram quantities of RNA
Genina Troutman Ian Miller Jeff Mueller Mike
The SYBR Gold nucleic acid gel stain (Molecular Probes), combined with Stratagenes Eagle Eye II still video system, allows the integrity of only 250 ng of poly(A)+ RNA to be effectively assessed.
In Stratagenes Custom Library Department, we often construct cDNA libraries from very limited amounts of RNA. Constructing a high-quality cDNA library requires high-quality poly(A)+ RNA. To practically assess the integrity of RNA, samples are run on an agarose gel, and the RNA is then visualized with a nucleic acid stain. Although ethidium bromide is commonly used to stain RNA gels, we found that staining with ethidium bromide often requires as much as 1 g of RNA to realize quality images from a variety of RNA samples. While we often have sufficient poly(A)+ RNA to make a library (ideally 5 g), in some cases the amount of poly(A)+ RNA is barely enough for cDNA synthesis even when using less than 1 g for a gel.
Various tissues or cells were collected, and total RNA was prepared by the acid guanidinium-phenol-chloroform extraction method.1 Poly(A)+ RNA was enriched by oligo(dT) selection.2 The RNA concentration was spectrophotometrically determined based on absorbance at 260 nm measured on a Beckman model DU640B spectrophotometer. A 250-ng aliquot (1 l) of each RNA sample was added to 5 l of loading buffer (72 l of formamide, 26 l of formaldehyde, 26 l of water, 16 l of 10X MOPS, 8 l of glycerol, and 1 l of saturated bromophenol blue). The samples were heated for 2 minutes at 65C and loaded onto a 1.25% SeaKem Gold agarose gel (Reliant RNA gel system, FMC). The gel was run at 100 V for one hour in 1X MOPS, then stained for one hour with SYBR gold 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.
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 run on two identical gels and stained with either SYBR Gold or ethidium bromide. When total RNA was stained with SYBR Gold, the ribosomal RNA bands were clearly visible when as little as 10 ng of total RNA was run on the gel, and the Eagle Eye II image integrated at 5 frames at 1/30 of a second (Figure 2, Panel A). But, when total RNA was stained with ethidium bromide, a minimum of 50 to 125 ng of total RNA was required for detectin, even when the image was integrated at 30 frames at 1/30 of a second (Figure 2 Panel B).
Use the SYBR Gold stain combined with the Eagle Eye II imaging system to clearly visualize 250 ng of poly(A)+ RNA or less on a gel. When the amount of sample RNA is the limiting factor for making cDNA libraries, this combined procedure provides a vast improvement over the 1 g of RNA often required for ethidium bromide staining. Hence, it is now possible to run an aliquot of every RNA sample on a gel, even when the amount of available poly(A)+ RNA is limited, and still have enough RNA available for cDNA synthesis.
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