Power sybr green kit

Total RNA was extracted from renal tissue specimens and cultured cells using previously described methods [39 (link), 40 (link)]. The Power SYBR Green kit (Invitrogen) and Bio-Rad CFX96 Real-Time system (Bio-Rad, Pleasanton, CA, USA) were used for qRT-PCR. The sequences of PCR primers are shown in the Supplementary Methods and Materials.

cDNA was synthesized from lens cell RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA and used for real-time quantitative PCR (qRT-PCR) conducted using the Power SYBR Green kit (Invitrogen Life Technology, Carlsbad, CA, USA) using the gene-specific primers shown in Supplementary Table S1. Three biological replicates were analyzed with 2 technical replicates each for each experimental condition and fold-change calculated using the ΔΔCT-method with GAPDH as the housekeeping gene. Statistical significance was determined using a Student’s two sample t-test.

Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA. Primers for IL-17, claudin-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by Gencopoia (Shanghai, China), and were as follows: claudin-1 forward 5′-GGAGACAGTTGAGTTC-3′ and reverse 5′-TAAGGCAGGATTATATTGAGTT-3′; murine IL-17 forward 5′-TGTAAGCCTAAGGAAGTC-3′and reverse 5′-GCAATCATAAGAGTAGTCA-3′; and murine GAPDH forward 5′-AGTGGCAAAGTGGAGATT-3′ and reverse 5′-GTGGAGTCACTGGAACA-3′. The Power SYBR Green kit (Invitrogen) and Bio-Rad CFX96 Real-Time system (Bio-Rad, Pleasanton, CA, USA) were used for performing qRT-PCR. Data were normalized to GAPDH expression.

Total RNA extraction was performed by manual extraction. Chloroform totaling 0.2 mL was added to 1 mL of TRIzol reagent (Thermo Fisher Scientific, Waltham, Massachusetts, United States), shaken vigorously for 15 s, and incubated at room temperature for 3 min. Samples were centrifuged at 12,000× g for 15 min at 4 °C. After collection, the upper phase was mixed with isopropyl alcohol and centrifuged at 12,000× g for 10 min at 4 °C. The sediment was washed with 75% ethanol and air-dried for approximately 30 min. Purified RNA was dissolved in 30 μL of RNase-free water and stored at −80 °C. About 500 ng of RNA was purified and subjected to reverse transcription using the cDNA with High-Capacity Reverse Transcription kit (Thermo Fisher Scientific, Waltham, USA). Real-time PCR was performed on an Applied Biosystems^® 7500 Fast Real-Time PCR System (Life Technologies; Carlsbad, CA, USA). qPCR analysis was conducted using a Power SYBR green kit (Thermo Fisher Scientific, Waltham, MA, USA) and the following cycles: 95 °C for 10 min, followed by 95 °C for 15 s and 58 °C for 1 min for 40 cycles. The sequences of primers are reported in Table 2. The relative difference of CLU and OCN gene expression between OP and CTR subjects was calculated using 2^−ΔΔCT method and normalized to GAPDH, β2-microglobulin (B2M) and β-actin levels as the internal control.