Cfx manager 3.1 software system

qPCR was performed using SYBRGreen^R and a Bio-Rad iQ iCycler Detection System. Primers were as follows: human SK-1: forward: 5′-GCT TCC TTG AAC CAT TAT G-3′; reverse: 5′-TCT CTA GGT CCA CAT CAG-3′; human CTGF: forward: 5′-TGC CTG CCA TTA CAA CTG TCC-3′, reverse: 5′-GCC ATG TCT CCG TAC ATC TTC C-3′; human fibronectin: forward: 5′-CGA AAT CAC AGC CAG TAG-3′; reverse: 5′-ATC ACA TCC ACA CGG TAG-3′; human Spns2: forward: 5′-ACT TTG GGG TCA AGG ACC GA-3′; reverse: 5′-AAT CAC CTT CCT GTT GAA GCG-3′; human MCP-1: forward: 5′-CCC CAG TCA CCT GCT GTT AT-3′; reverse: 5′-TGG AAT CCT GAA CCC ACT TC-3′; human aquaporin 1: forward: 5′-TGG ACA CCT CCT GGC TAT TG-3′; reverse: 5′-GGG CCA GGA TGA AGT CGT AG-3′; 18S RNA: forward: 5′-CGA TTC CGT GGG TGG TGG TG-3′; reverse: 5′-CAT GCC AGA GTC TCG TTC GTT ATC-3′. 1 μg of total RNA isolated with TRIzol^R (Thermo Fisher Scientific) reagent was used for reverse transcriptase cDNA synthesis (First Strand Synthesis Kit, Thermo Fisher Scientific); a random hexamer primer was used for amplification. Real-time fluorescence from SYBR^R Green (Sigma Aldrich) was measured by the Bio-Rad CFX Manager 3.1 System Software. The fold induction values were obtained, according to the ΔΔC_t method, after normalization to the housekeeping gene 18S RNA.

We isolated total RNA from cultured HK2 cells using the RNeasy Plus Mini Kit (QIAGEN, catalogue 74,136). Total RNA (1 μg) of purified RNA was used to prepare cDNA through high‐capacity RNA to cDNA kit (Applied Biosystems, catalogue 4,368,814). For candidate gene expressions, we used the fluorescent dye SYBR Green methodology and CFX96 Touch Real‐Time PCR machine (Bio‐Rad). We purchased the bioinformatically validated primer sets (QuantiTect Primer Assays, QIAGEN, or IDT) for use in SYBR Green‐based RT‐PCR and measured real‐time fluorescence from SYBR Green (Applied Biosystems) by the Bio‐Rad CFX Manager 3.1 System Software. We normalized gene expression to the endogenous controls (β‐actin) and estimated comparative CT values. We calculated relative gene expression through 2^–(ΔΔCT) method and expressed in arbitrary units (a.u.) relative to paired controls. We generated heatmaps of gene expressions by using the ‘Heatmapper’ web server.²⁷