Transcriptional Analysis of HIF-1α, AMPK and CS Using RT-qPCR

We used RT-qPCR to determine the transcript levels of HIF-1α, AMPK and CS. qPCR was performed in a semi-quantitative form using an ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA). qPCR reaction contained SYBR Select Master Mix (Thermo Scientific, Waltham, MA, USA), cDNA as template and a pair of specific primers (HIF1α-F: 5′-CAG TCG ACA CAG CCT GGA T-3′ and HIF1α-R: 5′-TGG CAA GCA TCC TGT ACT GT-3′; AMPK-F: 5′-CAG GCC ATA CCC TTG ATG AAT-3′ and AMPK-R: 5′-TTC TTC CTT CGT ACA CGC AAA T-3′; CS-F: 5′- ACC TGT CAG CGA GAG TTT GC-3′ and CS-R: 5′- CCC AAA CAG GAC CGT GTA GT-3′. Validation curves were run using 18S rRNA, which was selected as endogenous control for all analyzed genes (18S-F: 5′-TAC CGC AGC TAG GAA TAA TGG-3′ and 18S-R: 5′- CGT CTT CGA ACC TCC GAC TT-3′).

PCR reactions were performed in 96-well reaction plates using the recommended parameters (10 min at 95°C, 40 cycles of 95°C for 15 s, and 60°C for 1 min.). Each PCR reaction was performed by triplicate and two non-template controls were included. Data were analyzed with Sequence Detection Software v 1.3.1 (Thermo Scientific, Waltham, MA, USA) to establish the PCR cycle at which the fluorescence exceeded a set of cycle threshold (Ct) for each sample. Comparative 2^−ΔΔCt method was used for target gene expression analysis (21). After normalization using the 18S rRNA housekeeping gene, normalized data of AMPK, HIF-1α, and CS expression from tumor and fibroblast cells were compared with the average of normalized data from same genes, expressed in tumor and fibroblast cells cultured in neutral pH media under normoxia. This condition was set as the value of 1 and was used as calibrator to compare data from the acidic conditions under normoxia or hypoxia.