Tb green premix ex taqtm kit

TRIzol reagent was used to extract the total RNA from cells and exosomes (Invitrogen, Carlsbad, CA, USA). Hairpin-itTM miRNA qPCR Quantitation Kit (GenePharma, China) and PrimeScript RT reagent Kit (Takara, Japan) were used to create the cDNA for miRNA and mRNA, respectively. The TB Green® Premix Ex TaqTM kit was then used to carry out the qRT-PCR assay (Takara, Japan). The expression levels of mRNA and miRNA were compared to GAPDH and U6, respectively. The 2 − ΔΔCT method was used to compute the relative expression.

The RNA was collected by using the Takara MiniBEST Universal RNA Extraction Kit. The total mRNA was extracted from HNSCC cell lines. The Reverse Transcription Mix and PrimeScript RT Master Mix were also purchased from the Takara company, and the experimental protocols followed the instruction. The purity and concentration of the RNA were assessed by using a NanoDrop 2000/2000C spectrophotometer at wavelengths of 260/280 nm. A PrimeScript™ RT Reagent Kit (TaKaRa Biotechnology) was used to reverse transcribe RNA into cDNA. The resultant cDNA was used as a template in a TB Green® Premix Ex TaqTM Kit (TaKaRa Biotechnology) master mix, and qPCR reactions were performed on a StepOnePlusTM Real-Time PCR System. The homo UBE2C forward primer was 5’-GACCTGAGGTATAAGCTCTCGC-3’ and the reverse primer 5’-TTACCCTGGGTGTCCACGTT-3’. The homo NOX4 forward primer was 5’-CAGATGTTGGGGCTAGGATTG-3’ and the reverse primer 5’-GAGTGTTCGGCACATGGGTA-3’. The homo NOX4 forward primer was 5’-ATTTCCGTGGCTGGTACATTAG-3’ and the reverse primer 5’-ATGGCTGGTTGTTCGTCATCC-3’. The GAPDH forward primer is 5’- GGAGCGAGATCCCTCCAAAAT-3’ and the reverse is 5’- GGCTGTTGTCATACTTCTCATGG-3’.

RNA was extracted from renal tubular epithelial cells after exposure to hypoxic conditions for 24 h followed by 3, 6, and 9 h of reoxygenation for measurement of HMOX1 and miR-3587 expression. TRIzol (RNAiso Plus, TaKaRa, Japan, 108-95-2) was used to extract the RNA from the samples, and a NanoDrop 2000 spectrophotometer (Thermo Fisher) was used to assess RNA concentrations and purity. The EasyScript One-Step gDNA Removal and cDNA Synthesis Supermix (TransGen Biotech, AE311-02) were used to prepare cDNA using a StepOnePlus analyzer Real-Time PCR system (Applied Biosystems, United States). All qPCR analyses were performed using a TB Green PreMix Ex Taq TM kit (TaKaRa, RR420A) and an Applied Biosystems 7500 Real-Time PCR instrument. The primer sequences were as follows: β-Actin, positive: 5′-CTA TGA GGG TTA CGC GCT CC-3′ and reverse: 5′-ATG TCA CGC ACG ATT TCC CT-3'; HMOX1, positive: 5′-CAG AAG AGG CTA AGA CCG CC-3′ and reverse: 5′-TTG GTG AGG GAAA ATG TGC CA-3′. U6 and miR-3587 primers were synthesized by RiboBio (Guangzhou, China).

The total mRNAs were extracted from HIOEC and HNSCC cells lines. A NanoDrop 2000/2000C spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) was used to assess the RNA purity and concentration at wavelengths of 260/280 nm. The RNA was transcribed into cDNA using a PrimeScript™ RT Reagent Kit (Takara Biotechnology, Dalian, China). A TB Green® Premix Ex TaqTM Kit (Takara Biotechnology) master mix was used to perform the qPCR reactions using the cDNAs as templates on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) expression was detected as an internal control. The primers used for qPCR of human genes were:

Total RNA of the mouse lung tissue was isolated using Trizol (Thermofisher) according to the manufacturer’s instructions. Primer Script RT reagent kit (Takara, Japan) was used to synthesize complementary DNA, and real-time PCR was run on a Quant Studio 1 real-time PCR system (Thermofisher, United States) using the TB Green Premix Ex Taq^TM kit (Takara, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is stably expressed in the lung tissue during the process of pulmonary fibrosis. Therefore, it serves as a reliable endogenous control in the reverse transcription-polymerase chain reaction (Loitsch et al., 1999 (link); Cruz-Bermúdez et al., 2019 (link)). The 2^−△△Ct method was employed to assess relative expression levels of genes of interest. All of the data were presented as mean ± standard error of mean (SEM) and analyzed using the GraphPad PRISM8 software (United States). Two-tailed Student’s t test was used to compare between the two groups. p < 0.001 was considered statistically significant. Sequences of the primers could be found in Table 2.

Total RNA was obtained using an Axygen RNA Miniprep Kit (Axygen, USA) in accordance with the manufacturers’ instructions. A NanoDrop 2000/2000C spectrophotometer was used to test the RNA purity and concentration at wavelengths of 260 nm or 280 nm. A PrimeScript™ RT Reagent Kit (TaKaRa Biotechnology) was used to reverse transcribe 2 μg of the extracted RNA into cDNA. The resultant cDNA was used as a template in the TB Green^® Premix Ex TaqTM Kit (TaKaRa Biotechnology) master mix and qPCR reactions were performed on a StepOnePlusTM Real-Time PCR System. The following human primer sets were used: human APBB2: forward, 5-ATGGGACTGCGGAAGAGAAA-3 and reverse, 5-GCCCCTGTTTTCGGATGATC-3; human GAPDH: forward, 5-CCAGAACATCATCCCTGCCT-3 and reverse, 5-CCTGCTTCACCACCTTCTTG-3.

Cells were cross-linked with 1% Formaldehyde Solution (Sigma-Aldrich) for 15 min at 37 °C, quenched by 125 mM Glycine (Sangon Biotech, Shanghai, China) for 5 min at room temperature, and sonicated to shear DNA. CHIP assay was performed with CHIP Assay Kit (Beyotime) according to the manufacturer’s protocol. Chromatin fragments were precipitated with anti-AR (#51533, CST, 1:100 dilution) or a control non-immune IgG (#7074, CST, 1:100 dilution). The precipitate was eluted and analyzed by QPCR using TB Green Premix Ex TaqTM kit (Taka ra) and the Applied Biosystems QuantStudio instrument (Thermo Fisher). The primers of CHIP-QPCR were listed in Table S2.