Kapa sybr fast qpcr kit master mix universal kk4602

RNA extraction was performed using RNeasy Mini Kit from Qiagen by following the manufacturer's instructions. We used 500 ng of RNA for reverse transcription with SuperScript II Reverse Transcriptase from ThermoFisher Scientific. The resulting cDNA was used for qRT-PCR (Rotor-Gene Q from Qiagen). qRT-PCR were set up in triplicates with KAPA SYBR FAST qPCR Kit Master Mix Universal KK4602 from Kapa Biosystems. Relative gene expression and fold changes were determined using the 2^−ΔΔCT method using GAPDH as an internal control [53 (link)]. Primer sequences were: human CAIX forward GGG TGT CAT CTG GAC TGT GTT, human CAIX reverse CTT CTG TGC TGC CTT CTC ATC, human GAPDH forward CCA TGG GGA AGG TGA AGG TC, human GAPDH reverse ACG TAC TCA GCG CCA GCA TC, mouse CAIX forward GCT GTC CCA TTT GGA AGA AA, mouse CAIX reverse GGA AGG AAG CCT CAA TCG TT, mouse GAPDH forward AAG AGG GAT GCT GCC CTT A, mouse GAPDH reverse TTG TCT ACG GGA CGA GGA AA.

RNA extraction was performed using RNeasy Mini Kit from Qiagen by following the manufacturer’s instructions. We used 500 ng of RNA for reverse transcription with SuperScript II Reverse Transcriptase from ThermoFisher Scientific. The resulting cDNA was used for qRT-PCR (Rotor-Gene Q from Qiagen). qRT-PCR were set up in triplicates with KAPA SYBR FAST qPCR Kit Master Mix Universal KK4602 from Kapa Biosystems. The relative expression levels of the target gene mRNAs were calculated by the comparative C_T method (relative expression = 2^−ΔCT) using cyclophilin as an internal control. Primer sequences were: human VEGFR2 forward ATC CCT GTG GAT CTG AAA CG, human VEGFR2 reverse CCA AGA ACT CCA TGC CCT TA, human VEGFA forward CCT CCG AAA CCA TGA ACT TT, human VEGFA reverse ATG ATT CTG CCC TCC TCC TT, human cyclophilin forward ACC GTG TTC TTC GAC ATT GC, human cyclophilin reverse TTA TGG CGT GTG AAG TCA CC.