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

ected by capillary electrophoresis in an ABI Prism 310 Genetic Analyzer under denaturing conditions. One microliter of each PCR reaction or 1 ml of a mixture of PCR reactions was combined with 9 ml formamide and 0.51 ml of ROX 1000 size marker. Samples were then heated to 94 C for 5 minutes and flash-cooled in an ice slurry. Samples were loaded onto the machine and electrophoresed at 15 kV, 60 C for 35-45 minutes using POP4 polymer and filter set D. The default injection parameters of 15 kV, 5 seconds were used. The fragment analysis software GeneScan (Applied Biosystems, Inc., Foster City, CA,) was used to determine peak sizes in base pairs and peak heights which were used to calculate NT/CT ratios. No size correction was performed since each DNA molecule was tagged with one fluorescent marker from one labeled primer. The area under the curve values for each NT and CT was used for calculations.
Statistical analysis
All statistical analyses were conducted using SAS version 6.11 (SAS Institute, Cary, NC). A GLM test followed by Duncan analysis was used to test for significant differences in gene expression values obtained by the three methods of detection within the same cDNA. Differences were considered statistically significant if the P value was less than 0.05.
Results

Comparison of three methods of electrophoresis
Gene expression values for 15 genes were obtained by three methods of electrophoresis from the same cDNA, as described. Figure 1 on page 5 depicts representative results obtained by each of the three electrophoresis methods. The agarose gel shows the familiar band pattern (figure 1A). Although some of the bands are distorted a good resolution and quantification can be obtain
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