Brilliant 3 ultra fast sybr qpcr mastermix kit

RNA was extracted using Trizol-chloroform and in-column DNase digestion (Qiagen RNeasy Mini Kit). RNA concentrations and purity were determined using Nanodrop (ND-1000 Spectrophotometer, Thermo Fisher Scientific). 200 ng RNA was transcribed into cDNA using the AffinityScript multi-temp cDNA synthesis kit (Agilent Technologies, Cheshire, UK) as per manufacturer’s instructions. qRT-PCR was performed using primers described in Supplementary Table 2 with the Brilliant III Ultra-fast SYBR QPCR mastermix kit (Agilent Technologies) as per manufacturer’s instructions. mRNA expression for target genes was normalised using the mean (for one housekeeping (HK) gene) or the geometric mean (for more than one HK genes) of the following HK genes: beta-actin (ACTB), TATA-binding protein (TBP), beta-tubulin (TUBB). The ΔΔCt value was calculated as follows: $$\mathrm{\Delta }\mathrm{\Delta }\mathrm{Ct}=2^{-(\mathbf{Mean}\mathbf{Ct}[\mathrm{target}\mathrm{gene}]-(\mathbf{Geo})\mathbf{Mean}\mathbf{Ct}[\mathrm{HK}\mathrm{gene}(s)]}.$$

RNA was extracted from podocytes using the RNeasy miniprep kit (Qiagen, Manchester, UK), following the manufacturer’s instructions. The RNA concentration was determined by Nanodrop (Nanodrop 1000, ThermoScientific) at 260 nm, purity was assessed using the 260/280 nm ratio, and 200 ng RNA was transcribed into cDNA using the AffinityScript multitemp cDNA synthesis kit (Agilent Technologies, Cheshire, UK) following the manufacturer’s instructions. qRT-PCR was performed using the primers described in (Table 1) with the Brilliant III Ultra-fast SYBR QPCR master mix kit (Agilent Technologies) following the manufacturer’s instructions. The geometric mean of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein Zeta (YWHAZ), TATA-box-binding protein (TBP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference control for normalization.