RT-PCR

Total RNA was extracted from cultured cells using the RNeasy kit (Qiagen Inc., Mississauga, ON) according to the manufacturer's procedure. Complementary DNA was transcribed from 1.5 μg total RNA using a First Strand cDNA synthesis kit (Amersham, Oakville, Ont., Canada) and used as a template for polymerase chain reaction (PCR). All PCR primers span introns in order to detect specific mRNA sequences. The forward and reverse primers used were as follows: PBX1 (NM_002585): 5'-CCACGTGATGAATCTCCTGCGAGAG-3' and 5'-TCACTGTATCCTCCTGTCTGGCTGA-3', PBX2 (NM_002586): 5'-CTGGTTTGGCAACAAGAGGATTCGC-3' and 5'-TGGAGGTATCAGAGTGAACACTCCC-3' and MEIS1 (BC043503): 5'-AAGGTGATGGCTTGGACAA-3' and 5'-GGCTGCACTATTCTTCTCCG-3'. The expected size of PCR products using these sets of primers are 627 bp for PBX1a, 414 bp for PBX1b, 411 bp for PBX2, and 259 bp for MEIS1. The amplification reaction was carried out in the linear range of the logarithmic phase unless otherwise specified. Each cycle consisted of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, extension at 72°C for 60 s and a final extension at 72°C for 5 min in a DNA thermal cycler (Mini cyclerTM, PTC-100 TM, MJ-Research, Bio-Rad). PCR products were verified by sequence analysis.