2 µg total lung RNA of each animal from each treatment group at each time point was reverse transcribed in a volume of 20 µl using the following program:37°C for 15 min and 85°C for 5 s. 2 µl of cDNA was used in a 25 µl realtime PCR reaction volume.
The quantitative PCR reaction was carried out using the following program on Applied Biosystems 7900HT system (Applied Biosystems, Foster City, CA): 10 minutes at 95°C to activate HotStart DNA polymerase, followed by 40 cycles at 95°C for 15 seconds and at 60°C for one minute.
PCR was performed on an ABI-9700 using the following program.
5. Perform ligation on a thermal cycler from step 4 using the following program in Table 2.
The intron sequences were also scanned for microsatellites with Tandem Repeats Finder version 4.04 [ 49] using the following program parameters: 2 7 7 80 10 50 6.
A PCR thermocycler was run using the following program parameters: 95°C for 30 s, 95°C for 30 s, 18 cycles (54°C for 1 min, 67°C for 20 min, 94°C for 1 min, 55°C for 1 min, 72°C for 10 min).
The PCR reaction was carried out on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) using the following program, stage 1, 50°C for 2 min, stage 2, 95°C for 10 min, stage 3, 95°C for 15 s and 60°C for 1 min, for 40 cycles and, stage 4, 95°C for 15 s, 60°C for 15 s and 95°C for 15 s.
The apmplification was performed in a DNA Thermal Cycler (Perkin Elmer/Cetus Instruments), using the following program set: 95°C for 30 sec., 55°C for 30 sec., 72°C for 1 min. for a total number of 30 cycles plus an additional 9 min. at 72°C.
ERIC-PCR was performed briefly using the following program parameters: denaturation at 94°C for 1 s, annealing at 52°C for 10 s, and extension at 72°C for 35 s for 30 cycles, followed by a final extension at 72°C for 4 min. Amplicons were separated on a 1.5% agarose gel containing ethidium bromide (5 μg/mL) at 60 V for 3 hours.
qRT-PCR reaction used the following program: one cycle of 95°C for 5 min, followed by 44 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 45 s and extension at 72°C for 5 min. Ribosomal protein S7 was used as the standard for expression normalization for each gene and each age group.
