Amplification of cDNA by polymerase chain reaction (PCR) was performed using an annealing temperature and number of cycles optimized for each gene. For each PCR reaction, the number of cycles used was optimized so that the amplification process was carried out within the exponential (linear) range as demonstrated in Figure 1. To perform a semi-quantitative analysis of samples, serial dilutions of cDNA were subjected to increasing PCR cycles in order to define the linear amplification range for each primer set.
Four serial 10-fold dilutions of known amounts of HOXC10, HOXC11, HOXD10, HOXD11, or G3PDH cDNA were amplified in triplicate to construct standard curves for each primer pair Twenty-five nanograms of each cDNA sample were then amplified at the optimal cycle number for each gene of interest. Coamplification of the transcript of interest with the internal, G3PDH, allowed comparison between samples. The PCR reaction was conducted in a total volume of 50 |xl containing 10 X PCR buffer and 10 |xM of each of 5′ and 3′ primers (95°C, 1 min; 65.7°C for HOXC10, 58.6°C for HOXC11, 65.7°C for HOXD10, and 66.2°C for HOXD11, 1 min; 72°C, 1.5 min). All the components for reverse transcription (RT)-PCR were purchased from Promega (Madison, WI) with the exception of the primers that were synthesized at the Yale University School of Medicine, Department of Pathology.