Semiquantitative Reverse Transcription-Polymerase Chain Reaction
QIAGEN RNAeasy kit (QIAGEN, Venlo, The Netherlands) was used to extract mRNA according to the manufacturer’s instructions. Reverse transcription was carried out with 2 |xg of sample in 20 |xl of reaction mixture containing 10 mM each of dATP dCTP, dGTP, and dTTP; 20 pmol oligo (dT); 40 u/^l of ribonuclease inhibitor; 10 u/^l of avian myeloblastosis virus-reverse transcriptase; and 5X AMV-RT buffer (42°C, 60 min; 95°C, 5 min: Eppendorf Mastercycler Gradient, Brinkmann, West-bury, NY). Primers that specifically amplified each of these genes were designed and tested; four pairs of primers were used for subsequent HOX amplification.
The primers used for amplification of G3PDH were as described by Apostolakos et al. The HOX primer sequences and product sizes are as follows: H0XC10, 5′-AAGACCTCAGACTCTCCTTCCAA-3′ and 5′-GAGAACAGAATGCTGTGTGTGAG-3′ (189 base pair [bp]); HOXC11, 5′-AATGTCTTGCTGCTCGGATTAG-3′ and 5′-TAACAC-CAGGTTGAAGGTACAGAA-3′ (225 bp); H0XD10, 5′-ATGTACATG-CCACCACCTAGC-3′ and 5′ -TTGCTGTGTAACAGGTTGCTCTA-3′ (192 bp); HOXD11, 5′ -TGTACCTGCCGGGCTGCGCCTACTATGTGG-3′ and 5′-GGCTGGACGTGCGGAGCCAGGTTGGAAGAGT-3′ (130 bp); G3PDH, 5′-GGTCGGAGTCAACGGATTTGGTCG-3′ and 5′-CTT-CCGACGCCTGCTTCACCAC-3′ (788 bp).