Cdna reverse transcription kit

The miRNA-9 mimics and inhibitors were transfected into macrophages RAW264.7. An appropriate amount of Trizol reagent was added to the treated cells to lyse the cells. After the chloroform reagent was added for 10 minutes, the solution was centrifuged at low temperature and high speed for 15 minutes to collect the supernatant. After adding an equal volume of isopropanol and mixing, the supernatant was allowed to stand at room temperature for 5 minutes, and then centrifuged at low temperature and high speed for 10 minutes to take the precipitate. The precipitate was washed with a pre-cooled 75% ethanol solution and dried naturally, added an appropriate amount of RNase-free ultrapure water to dissolve, and stored in a refrigerator at −80°C. Reverse transcription of complementary deoxyribonucleic acid (cDNA) was performed according to the instructions of the cDNA reverse transcription kit (Takara, Japan). The cDNA was undertaken as a template, and the target gene was quantified according to the instructions of the qPCR detection kit (Takara, Japan). With glyceraldehyde-3phosphate dehydrogenase (GAPDH) gene as an internal reference, the relative expression level of the target gene was calculated according to 2^−ΔΔCT [8 (link)].

Total RNA was extracted from NYG-treated cells using RNA isoplus reagent (Takara, Tokyo, Japan). Reverse transcription was performed using cDNA reverse transcription kit (Takara). Quantitative real-time PCR was performed with SYBR premix Ex Taq (Takara) with Light Cycler 480 real time PCR system (Roche, Basel, Switzerland). The primer sequence is as followed: VEGF 5’-CCTCCGAAACCATGAACTTT-3’ (forward) and 5’-TTCTTTGGTCTGCATTCACATT-3’ (reverse).

Total RNA from WT and KO mice testicular tissues were extracted by TRIzol reagent (15596026; Thermo Fisher). Then, 1 μg of total RNA was reverse transcribed using the cDNA Reverse Transcription kit (6110A; Takara). Quantitative Real-Time PCR was performed using TB Green™ Premix Ex Taq™ II (RR420A; Takara) on a LightCycler 96 Real-Time PCR System (Roche), according to the manufacturer’s instructions. All primers for genes are listed in Supplementary Table 1 and were synthesized by Genewiz (Suzhou, China). Relative abundance of mRNA expression was calculated with the 2^−ΔΔCt method and normalized to β-actin mRNA expression levels.

Cells were cultured with RPMI 1640 with 5% charcoal dextran-treated FBS for 5 days before treatment with R1881 (Sigma, catalogue no. R0908) alone or R1881 and AIL for 12 h. Total RNA was extracted using TRIzol (Takara, Japan) according to the manufacturer's instructions. One microgram of total RNA was used for complementary DNA synthesis using a cDNA reverse transcription kit (Takara, Japan). Real-time PCR was performed in triplicate using gene-specific primers on a Stratagene Mx3005P PCR system (Agilent Technologies) machine. The mRNA expression levels were normalized to β-actin expression or GAPDH. All analysis was performed using Microsoft Excel 2010 and GraphPad Prism 5 software. The gene-specific primers are listed in Supplementary Table 2.

Total RNA was isolated from frozen livers using Trizol (Invitrogen), according to the manufacturer’s instructions. RNA (1 μg) was reverse-transcribed using the cDNA Reverse Transcription Kit (Takara Bio Inc, Japan), according to the manufacturer’s protocols. Real-time quantitative PCR (RT-qPCR) was performed using an Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, USA) and Premix Ex Taq (Probe qPCR), according to the protocols provided by the manufacturer. Briefly, PCR was performed in a final volume of 20 μL containing 800 ng of cDNA, 0.4 μL of 0.4 mmol/L forward and reverse primers, 0.4 μL of 0.4 mmol/L fluorescence probe, 10 μL of 1.25 U/25μL Premix Ex Taq, and 0.4 μL of 25 μmol/L ROX Reference Dye. PCR reactions consisted of an initial denaturing step at 95°C for 30 s, followed by 45 cycles of 10 s at 94°C and 37 s at 60°C. Primers and probes are presented in Table 1. Results are presented as levels of expression relative to those of controls after normalization to β-actin and glyceraldehyde 3-phosphate dehydrogenase (GADPH) using the 2^-ΔΔCT method.

Total RNA was isolated from controls or transfected cells by the RNeasy kit (Qiagen, USA), and miRNA supplementary DNA (cDNA) was transcribed from total RNA using the cDNA reverse transcription kit (Takara Bio, Korea). Real-time PCR was performed by IQ SYBR Green Supermix (1708886, Bio-Rad, USA) in line with the supplier’s guidance. The primers below were applied: miR-194-5p forward 5′-CTAGTACCTAGAGGAACCTTTGAAGACTGTTACAGCTCAGCA-3′, reverse 5′-AGCTTGCTGAGCTGTAACAGTCTTCAAAGGTTCCTCTAGGTA-3′; GAPDH forward 5′-CCCACTCCTCCACCTTTGAC-3′, reverse 5′-CCACCACCCTGTTGCTGTAG-3′. GAPDH was used as a loading control, and the comparative gene manifestation was determined by the ΔΔCT method. The qPCR assays were conducted three times.