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

taRT-PCR
StaRT-PCR was performed using previously published protocols with G.E.N.E. system 1a gene expression kit (Gene Express National Enterprises, Inc., Huntsville, AL). Briefly, a master mixture containing buffer, MgCl2, dNTPs, cDNA from normal human bronchial epithelial cells (lot 17378, American Type Culture Collection, Rockville, MD), CT mixture from G.E.N.E. system 1a kit and Taq polymerase (Promega, Madison, WI) was prepared. Aliquots of this mixture were dispensed into tubes containing genespecific primers and cycled either in a Rapidcycler (Idaho Technology, Inc., Idaho Falls, ID) or PTC- 100 block thermal cycler with heated lid (MJ Research, Inc., Incline Village, NV) for 35 cycles. In each protocol the denaturation temperature was 94 C, the annealing temperature was 58 C, and the elongation temperature was 72 C. All StaRT-PCR reagents had been optimized for the same PCR conditions. NT and CT PCR products from the Rapidcycler were separated and evaluated on an agarose gel or in the Agilent 2100 bioanalyzer as described below. Primer pairs used for amplification in the PTC-100 block thermal cycler consisted of one fluorescently labeled primer and one unlabeled primer for each gene. These NT and CT PCR products were separated and analyzed in an ABI Prism 310 Genetic Analyzer.
Electrophoresis and quantitation
Agilent 2100 bioanalyzer
For analysis with the Agilent 2100 bioanalyzer, the DNA 7500 LabChip kit was used. Following amplification in the Rapidcycler, 1 l of each 10 l PCR reaction was loaded into a well of a chip prepared according to protocol supplied with the DNA 7500 LabChip kit. Briefly, 9 l gel-dye matrix was loaded into the chip in one well and the chips were pressurized for 30 seconds. Two additional wells were filled with geld
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