Quantifying RANK Isoform Expression in Endometrial Cancer

OriGene endometrial cancer array supplemented plates were loaded into each well with 30 µL of a master mix stock solution containing 2× SYBR green mix (Life technologies, Carlsbad, CA, USA), water, and specific primers at 0.05 µm final concentration. The sequence of primers for β-actin, RANK (TNFRSF11A) and its variant RANK isoforms lacking exon 9 (TNFRSF11A_Δ9), exons 8–9 (TNFRSF11A_Δ8,9), and exons 7–9 (TNFRSF11A_Δ7,8,9), has been previously described [20 (link)]. Real-time PCR was performed using an ABI PRISM 7500 Sequence Detection System (Perkin Elmer Corp., Norwalk, CT, USA) according to the manufacturer instructions with a heated lid (105 °C), an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative expression level of RANK was calculated with the comparative 2^ΔΔCt method, where ΔC_t = C_t(target) − C_t(control), ΔΔC_t = C_t(target) − C_{t(calibrator)} and all samples were normalized to the β-actin gene.
In order to quantify alterations in profiling pattern of RANK isoforms associated with cancer parameters, the expression of each RANK isoform per case was normalized by dividing its specific 2^ΔΔCt value by the summation of 2^ΔΔCt values from the four RANK isoforms. Results were expressed as percentage.