Ab high capacity rna to cdna kit

RNA was extracted from kidneys using an RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified by spectrophotometry. cDNA was prepared by reverse transcription of 1 µg of RNA using the AB High Capacity RNA-to-cDNA Kit (Life Technologies, Grand Island, NY). Quantitative real-time PCR was performed with PerfeCTa SYBR Green Fastmix (Quanta, Surrey, UK) and the Mx3000 Real-Time PCR system (Stratagene, La Jolla, CA). mRNA levels were calculated using the ΔΔCt method, using 18S as a reference gene. Primers were designed based on published sequences for 18S, Ang1, Ang2, alpha smooth muscle actin (αSMA), and PDGF beta receptor (PDGFRβ) and are listed in Table 1.

RNA was extracted using QiaShredder lysis buffer and purified using RNeasy spin columns from Qiagen. Purified RNA (900 ng) was used as a template to produce cDNA using AB high Capacity RNA-to-cDNA kit (Life Technologies) according to the manufacturer’s protocol on a Bio-Rad C1000 Thermo Cycler. Approximately 20 ng of cDNA was used for quantitative real-time PCR (qPCR) experiments using Eppendorf plates and AB Power-SYBR mix (20 μL final reaction volume) on an Eppendorf Mastercycler ep realplex S. Primers were obtained from IDT and used at a final concentration of 200 nM. The following cycle conditions were used for all genes: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 54 °C for 60 s with a final extension step of 95 °C for 15 s and 54 °C for 60 s. The ΔΔCt method was used to calculate fold changes in RNA levels compared to vehicle treated cells after normalization to a reference gene, U36-B. The primer sets used were reported previously [13 (link)] as follows: human U36B-F, 5′-CGAGGGCACCTGGAAAAC-3′; human U36B-R, 5′-CACATTCCCCCGGATATGA-3′; human GRN-F, 5′-CAGGGACTTCCAGTTGCTGC-3′; human GRN-R, 5′-GCAGCAGTGATGGCCATCC-3′.