Iqtm sybr green supermix buffer

Total RNA of normal and tumor tissue were reverse transcribed with the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA), following the manufacturer's instructions. Quantitative real-time polymerase chain reactions (PCR) were performed using iQTM SYBR Green Supermix buffer (6 mM MgCl₂, dNTPs, iTaq DNA polymerase, SYBR Green I, fluorescein, and stabilizers) (BIO-RAD Laboratories, Hercules, CA). The following primers were used for real-time PCR: 5′-GATCCTCCTGGCCAACTTCT-3′ and 5′-GTTGGTTGGGCGATTTCCTT-3′ for GPI/AMF; 5′-AATCTGGCACCACACCTTCT-3′ and 5′-AGCCTGGATAGCAACGTACA-3′ for β-actin. Quantification of the mRNA levels was performed on a MiniOpticon real-time PCR detection system (BIO-RAD Laboratories). In the PCR reactions, the following protocol was used: activation of the polymerase at 95°C for 3 minutes, followed by 45 cycles at 95°C for 10 seconds, 60°C for 30 seconds. Melting curves were generated through 60 additional cycles (65°C for 5 seconds with an increment of 0.5°C/cycle). Gene expression results were obtained as mean Ct (threshold cycle) values of triplicate samples. Expression was determined using the 2^−ΔΔCt method. Expression values were normalized to β-actin.

Total RNA of transfected normal and tumor kidney cells was reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems Foster City, CA, USA), following the manufacturer's instructions. Quantitative real-time polymerase chain reactions (PCR) were performed using iQTM SYBR Green Supermix buffer (6mMMgCl2, dNTPs, iTaq DNA polymerase, SYBR Green I, fluorescein and stabilizers) (BIO-RAD Laboratories, Hercules, CA, USA). The primers used for G6PDH were 5′-GAGGCTGCAGTTCCATGATG-3′ and 5-'GACTCCTCGGGGTTGAAGAA-3′. Quantification of the mRNA levels was performed on a MiniOpticon Real-Time PCR detection system (BIO-RAD Laboratories). In the PCR reactions the following protocol was used: activation of the polymerase 95°C for 3 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s. Melting curves were generated through 60 additional cycles (65°C for 5 s with an increment of 0.5°C/cycle). Gene expression results were obtained as average Ct (threshold cycle) values of triplicate samples. Expression was determined using the 2^−ΔΔCt method. Expression values were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).