Primescript rt master mix perfect real time kit

Total RNA was extracted from HTMCs using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and reverse transcribed into cDNA using the PrimeScript™ RT Master Mix (Perfect Real Time) kit (Takara Biotechnology Co., Ltd., Dalian, China), then qPCR was performed using the SYBR ExScript RT-PCR kit (Takara Biotechnology Co., Ltd.) on an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The thermocycling profiles are shown in Table I. The relative expression of PTEN in each sample was normalized to the level of GAPDH using the 2^−ΔΔCq method. The expression of miR-17-5p was examined using the Hairpin-it™ miRNAs qPCR Quantitation Kit (GenePharma Co., Ltd.) according to the manufacturer's protocol, and U6 (RNU6B; GenePharma Co., Ltd.) was used for normalization. Primers were synthesized by GenScript (Nanjing, China), and the sequences of the primers are shown in Table II.

TRIzol® reagent was obtained from Nanjing KeyGen Biotech, Co., Ltd. (KGA1203; Nanjing, China); a PrimeScript® RT Master Mix Perfect Real Time kit (DRR036A) and SYBR® Premix Ex Taq™ (DRR420A) were purchased from Takara Biotechnology Co., Ltd. (DRR036A; Dalian, China); polyclonal rabbit anti-mouse HO-1 and rabbit anti-mouse CSE antibodies were obtained from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China); anti-rabbit IgG was obtained from Fuzhou Maixin Biotechnology Development Co., Ltd. (MaxVision™2; Fuzhou, China); a Takara RNA polymerase chain reaction (PCR) kit (alfalfa mosaic virus) Ver. 3.0 was purchased from Takara Biotechnology Co., Ltd.; and CoPP and ZnPP were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Total RNA of each sample was isolated using TRIzol reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The quality and concentration of extracted RNAs were identified by measuring the absorbance at 260 nm. First-strand cDNAs were generated with the PrimeScript RT Master Mix Perfect Real Time kit (Takara Biotechnology Co., Ltd.) by mixing the components and incubate at 42°C for 1 h. All qPCRs were performed with SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) on an ABI PRISM 7500 Real-Time system (Thermo Fisher Scientific, Inc.). Initial denaturation took place at 95°C for 5 min, followed by annealing at 60°C for 30 sec, and a final extension at 72°C for 5 min. These conditions were cycled 40 times. The primers used are listed below, and GAPDH was included as the internal control. Each experiment was performed in triplicate at least three times. The primer sequences used are as follows: TRIM59 forward, 5′-TACGAGAGCAGCAGCTTGAA-3′; and reverse, 5′-ACGGGTTGAACCTCAGGAAG-3′; GAPDH forward, 5′-GTGGACATCCGCAAAGAC-3′; and reverse, 5′-AAAGGGTGTAACGCAACTA-3′. The sequence of the short hairpin RNAs against Trim59 were as follows: shTrim59#1, 5′-ACATTACAGGCAACCATTAAA-3′; shTrim59#2, 5′-TCCTCGTGTACTGCCATGCTCTCAT-3′; sh (negative control) NC: 5′-GGGTGAACTCACGTCAGAA-3′.

Total RNA was extracted from VSMCs using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, and then the RNA was reverse transcribed using the PrimeScript RT Master Mix Perfect Real Time kit (TaKaRa, Dalian, People’s Republic of China) to obtain the cDNA. Using the cDNA as the template, a real-time PCR assay was performed using the following pairs of primers: ERα forward, 5′-CGCTTTTGAACCAGCAGG-3′ and ERα reverse, 5′-TTCCCGAGGCTTTGGTGTG-3′; ERβ forward, 5′-ATCTGTCCAGCCACGAATCA-3′ and ERβ reverse, 5′-ATTAGCACCTCCATCCAGCA-3′; GPR30 forward, 5′-AGCTCAGGCTGTATGTGGCG-3′ and GPR30 reverse, 5′-TGCTCCGTGCTGTCTGGTAT-3′; and β-actin forward, 5′-AGGCCCCTCTGAACCCTAAG-3′ and β-actin reverse, 5′-CCAGAGGCATACAGGGACAAC-3′. The 20 µL real-time PCR reaction included 0.5 µL of cDNA template, 0.25 µL of Primer F, 0.25 µL of Primer R, 10 µL of RNase-free dH₂O, and 8 µL of 2.5× RealMasterMix (SYBR Green I). The reaction conditions included a predenaturation step at 95°C for 10 seconds, and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. After the reaction, the data were subjected to statistical analysis.

Total RNA from the human tissues and cultured cells were extracted using TRIzol solution (Takara Biotechnology Co., Ltd., Dalian, China) and quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). A 1 µg sample of mRNA was reverse transcribed using PrimeScript RT Master Mix (Perfect Real Time) kit (Takara Biotechnology Co., Ltd.) and qPCR was performed in an ABI PRISM 7900 Real Time system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.). The primers used were as follows: Tim-3, forward 5′-GCTACTACTTACAAGGTCCTCAG-3′ and reverse 5′-ATTCACATCCCTTTCATCAGTC-3′; GAPDH, forward 5′-GTGGACATCCGCAAAGAC-3′ and reverse 5′-AAAGGGTGTAACGCAACTA-3′. U Initial denaturation was performed at 95°C for 30 sec, and PCR by 40 cycles of 95°C for 5 sec and 60°C for 35 sec. All experiments were performed in triplicate at least three times. Values were calculated used the 2^−ΔΔCq method (25 (link))