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Improved Sensitivity for Staining RNA with SYBR Gold Stain


Eagle Eye II still video system provides quality images from nanogram quantities of RNA

Genina Troutman Ian Miller Jeff Mueller Mike Kobrin
Stratagene

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.

Methods

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
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