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Quantitative end-point RT-PCR gene,,,expression measurement using the,,,Agilent 2100 Bioanalyzer and,,,standardized RT-PCR

ye matrix and the remaining wells were loaded with 5 l each of molecular weight marker. One microliter of DNA ladder was loaded into a ladder well and 1 l of PCR product was loaded into each sample well. The chip was vortexed and placed into the Agilent 2100 bioanalyzer. DNA 7500 assay was run, which applies a current sequentially to each sample to separate products. DNA is detected by fluorescence of the intercalating dye in the gel-dye matrix. NT/CT ratios were calculated from the area under the curve for each PCR product and a size correction was made since an intercalating dye was used to detect DNA. The area under the curve values for each NT and CT was entered into the G.E.N.E., Inc. spread sheet. Alternatively, the concentration values, which are based on the areas, can be used to calculate the NT/CT ratio.
Agarose gel electrophoresis
Following amplification in the Rapidcycler, PCR products were loaded directly onto 4 % agarose gels (3:1 NuSieve: SeaKem) containing 0.5 g/ml ethidium bromide. Gels were electrophoresed for approximately one hour at 225 V and visualized with a Foto/Eclipse image analysis system (Fotodyne, Hartland, WI). Digital images were saved on a Power Mac 7100/66 computer and Collage software (Fotodyne) was employed for densitometric analysis. Quantification of gene expression was determined according to the G.E.N.E., Inc. manual. These mathematical steps are programmed as formulas into a spread sheet available directly or downloadable from G.E.N.E., Inc. so that only the raw data values for each NT and CT for each gene must be entered. The gene expression values then are calculated automatically.
Capillary electrophoresis
PCR products amplified with fluorescent primers in the PTC-100 block thermal cycler were det
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