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