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The quantitative real-time PCR was set up as follows: 10 ng of RNA was used as template for each real-time PCR reaction (10 µg reaction volume); primer pairs at 0.3 µM for GAPDH with Syber Green Master Mix (Applied Biosystems).
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Real-time PCR was set up with Supermix (Bio-Rad) containing syber green for 50 cycles with a three-step program.
Real-time PCR was set up with the respective cloning primers, and the fluorescent probe 5'-6FAM-ATGGACGGCTAGTGTAGGAGTCTGCCGA XT p (GenBank NM_000723; position: 79-52) and run at 94°C (3 min), and 40 cycles of 56.5°C, and 94°C (each 30 sec).
Real-time PCR was set up with the cloning primers and run at 94°C (3 min), and 40 cycles of 50°C (30 sec), 72°C (20 sec), and 94°C (20 sec).
Real-time PCR was set up using sybergreen-containing supermix (Bio-Rad) for 50 cycles with a three-step program (25 sec denaturation at 96°C, 30 sec annealing at 60°C, and 30 sec extension at 72°C).
Real-time PCR was set up with the respective cloning sense primer and antisense primers, and the fluorescent probe 5'-6FAM-CTCCAGTTCCAGTCTGGGAGATGTGGT XT p (GenBank NM_000723; 720-746) and run at 94°C (3 min), and 40 cycles of 53°C, and 94°C (each 30 sec).
The real-time PCRs were set up according to the manufacturer's instructions (SYBR Premix Ex Taq Kit, Takara Bio).
Real-time PCR was set up with the Stratagene Mx3000p (Agilent Technologies, Santa Clara, CA, USA) by using Brilliant® II SYBR Green QPCR Master Mix (Agilent Technologies).
For each DNA sample, real-time PCR was set up containing three primers (a specific primer for wild-type and APCmin and a common antisense primer; Table 2[ 7]) to detect each allele.
