Oligo dT-primed complementary DNA (cDNA) was synthesized from 1 μg total RNA, using a commercial reverse transcriptase enzyme (SuperScript III Reverse Transcriptase; Life Technologies Corp.), according to the manufacturer's procedure. The equivalent of 150 ng cDNA was used to PCR amplify transcripts expressed by the following genes implicated in apoptosis
28 : B-cell lymphoma protein-2 (
Bcl-2); Bcl-2 associated protein X (
Bax); cysteinyl aspartic acid-protease-3 (
Caspase-3); cellular oncogene FBJ murine osteosarcoma viral oncogene homolog (
c-Fos); cellular oncogene V-jun avian sarcoma virus 17 oncogene homolog (
c-Jun); mitogen-activated protein kinase-11 (
Mapk-11);
Mapk-12;
Mapk-13;
Mapk-14; and signal transducer and activator of transcription-3 (
Stat-3) genes. In murine tissues, the
Bcl-2 gene is expressed as two isoforms, whereas the
Bax gene is transcribed as a single mRNA transcript. Being related genes,
Bcl-2 and
Bax encode for transcripts that share a high sequence identity. To ensure cross-hybridization would not occur during the qPCR experiments, primers for these genes were carefully designed with specific 3′-ends, such that polymerization would only occur to amplify a specific product. All reactions used a commercial qPCR kit (SYBR Green PCR Master Mix; Applied Biosystems, Warrington, UK) and 5 μM final concentration of each forward and reverse primers (
Table). All primer pairs were designed to span an intron of the target gene to minimize genomic DNA (gDNA) contamination. All qPCR experiments were performed in triplicate using a commercial real-time PCR instrument (StepOnePlus Real-Time PCR System; Applied Biosystems) with the following settings: an initial denaturation step of 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds, an annealing temperature of either 55°C (all genes except
Bcl-2) or 60°C (for
Bcl-2) for 30 seconds, and extension at 72°C for 30 seconds. Values obtained for the target genes were normalized to the geometric mean of three housekeeping genes, namely acidic repeat protein (
Arp), beta-actin, and glyceraldehyde-3-phosphate dehydrogenase (
Gapdh). Primer sequences were designed using primer design software (Primer3 program; provided in the public domain at
http://frodo.wi.mit.edu/cgi-bin/primer3/)
29 and standard curve analysis was performed to ensure primer efficiencies were close to 100%.