Glass slide-based agarose gel electrophoresis for determining the efficiency of RNA amplification and cyanine dye incorporation during RNA labeling

cedure that allows for the quality control of amplification and cyanine dye labeling of aRNA in a single step. The implementation of such controls can avoid hybridization with targets having low specific activity and, in the case of two-color hybridizations, allows for corrections in targets with differences in their specific activity that otherwise could compromise normalization. In our experiment, 1.5 µg total RNA derived from a disease-free human esophagus obtained from our tumor bank were amplified by a T7-based protocol that was slightly modified from Wang et al. (3). After amplification, 3 µg aRNA were labeled with either Cy™3- or Cy5- (Amersham Biosciences, Piscataway, NJ, USA) αdCTP using random primer in two independent reactions. For analysis, the samples of labeled cDNA were mixed and fractionated through a 1% agarose gel that was mounted over a glass slide, and migration was performed along the longer axis of the slides using 1× TAE (40 mM Tris-acetate, 1 mM EDTA, pH 9.0) and 60 V. To cast the gel, 2.5-mm polycarbonate plastic strips are fixed along the edge of one standard microscope glass slide (75 × 25 × 1 mm), which creates the gel box. The gel box is then placed into an electrophoresis apparatus