Sybr green mix kit

Total RNA was isolated from GC-1 cells and testis samples using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and the concentration of RNA was determined by a DU800 UV/Vis Spectrophotometer (Beckman Coulter, CA, USA). Total cellular RNAs were reversed transcribed into cDNA using reverse transcription reagent kit (Takara Biotechnology, Dalian China). Real-time quantitative PCR was performed via a Applied Biosystems SYBR Green mix kit and the ABI 7900 Real-Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA). Relative miR-29a or TRPV4 mRNA expression were normalized to snRNA U6 (for miRNAs) or GAPDH (for mRNAs), respectively. The quantitative analysis was calculated by using 2^−ΔΔCt method (Rao et al., 2013 (link)). The primer sequences used are shown in Table 1.

RNA isolation was done from the clinical specimens, as well as PCa cells with the TRIzol^® reagent (Invitrogen). Afterwards, cDNA was generated from the RNA with the Takara RNA PCR kit (Takara Biotechnology). Next, qPCR was conducted on the ABI 7900 Real‐Time PCR platform by utilizing an Applied Biosystems SYBR Green mix kit (Applied Biosystems Life Technologies). Relative miR‐363‐3p or DKK3 mRNA expression was normalized to U6 (for miRNAs) or GAPDH (for mRNAs), respectively. miRNA or mRNA relative amounts of were computed with the 2^−∆∆Ct approach. Oligonucleotide sequences are given in Table 1.

Total RNA was extracted from T24 cells using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and the RNA purity was determined using a DU800 UV/Vis Spectrophotometer (Beckman Coulter, CA, USA). Subsequently, total cellular RNAs were reversed transcribed into cDNA using a reverse transcription reagent kit (Toyobo, Osaka, Japan). Real‐time quantitative PCR was performed via an Applied Biosystems SYBR Green Mix Kit and an ABI 7900 Real‐Time PCR System (Applied Biosystems Life Technologies, Foster City, CA, USA). The expression of NDE1 mRNA was normalised to GAPDH, respectively. The relative amount of mRNA was calculated using the 2‐∆∆Ct method. The primer sequences used are shown in Table 1.

Total RNA from the kidney cortex was used to synthesize cDNA using SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA). Quantitative PCR was carried out by using specific primers (Table 1). A SYBR Green Mix Kit (Applied Biosystems, Foster City, CA) and an ABI Prism 7500 Real-Time System (Applied Biosystems, Foster City, CA) were used for PCR. Relative expression levels were calculated with the 2^−ΔΔCt method.

Total RNA was isolated from the rat tissue from each group using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and the RNA concentration was obtained using a spectrophotometer. Single-stranded cDNA was synthesized using a cDNA synthesis kit (Takara, Kyoto, Japan) according to the instructions provided by the manufacturer. qPCR was performed with the Applied Biosystems SYBR Green mix kit (Applied Biosystems, Foster City, CA, USA) using the SLAN-96s Real-Time PCR system (Shanghai Hongshi Medical Technology Co., Ltd., Shanghai, China). The reaction composition contained: 2 μl cDNA, 12.5 μl 2X SYBR Green mix, 1 μl forward primer and 1 μl reverse primer and 8.5 μl ddH₂O in a final volume of 25 μl. The primers used were as follows: Bax forward, 5′-TGAACTGGACAACAACATGGAG-3′, and reverse, 5′-AGCAAAGTAGAAAAGGGCAACC-3′ (GenBank accession number NM_017059); Bcl-2 forward, 5′-TTTGATTTCTCCTGGCTGTCT-3′ and reverse, 5′-CTGATTTGACCATTTGCCTG-3′ (GenBank accession number NM_016993); PARP-1 forward 5′-TCTCCAATCGCTTCTACACCCT-3′ and reverse, 5′-TACTGCTGTCATCAGACCCACC-3′ (GenBank accession number NM_013063). β-actin was used as a housekeeping gene. The data are presented as a ratio of gene to β-actin mRNA [sense: 5′-TGCTATGTTGCCCTAGAC NM_017059 TTCG-3′ and antisense: 5′-GTTGGCATAGAGGTCTTTACGG-3′ (GenBank accession number NM_031144).