Ecodry premix random hexamers

Frozen tissues were pulverized using a stainless steel mortar and pestle (Cellcrusher) chilled in liquid nitrogen and homogenized in 700 μl QIAzol (79306, QIAGEN) with a Precellys CK28-R hard tissue homogenizing kit (P000916-LYSK0A, Bertin). Total RNA was extracted from homogenized tissues by using the miRNeasy Mini Kit (217994, QIAGEN), and the concentration and quality of RNA was determined by absorbance at 260/280 nm with a NanoDrop One Spectrophotometer (Thermo Scientific, USA). cDNA was prepared using RNA to cDNA EcoDry™ Premix-Random Hexamers (639546, Takara Bio, USA). Quantitative PCR was performed on reverse-transcribed cDNA using TaqMan™ Universal PCR Master mix (4304437, Applied Biosystem) to analyze mRNA expression levels. Relative expression of mRNA was determined by the ΔΔCt method.

Human monocytic cells THP-1 (TIB-202, ATCC) were maintained in Roswell Park Memorial Institute medium (RPMI 1640) (30-2001, ATCC) supplemented with 2-mercaptoethanol (31,350,010, Gibco) to a final concentration of 0.05 mM, fetal bovine serum (30-2020, ATCC) to a final concentration of 10% and penicillin–streptomycin-amphotericin B solution (PCS-999–002, ATCC) at a dilution of 1:1000. To assess macrophage activation, 1.5 × 10⁶ THP-1 cells were treated with activn A (5 ng/mL) or GDF11 (5 ng/mL) for 24 h, or activin B (50 ng/mL) for 6 h, in serum-free media. Prior to treatment, cells were growth-arrested overnight in serum free media. After treatment, cells were washed twice with ice-cold PBS and lysed with Buffer RLT plus (1,053,393, Qiagen). Total RNA was extracted with RNAeasy plus kit (74,134, Qiagen) according to the manufacturer’s instructions. cDNA was prepared using RNA-to-cDNA EcoDry™ Premix-Random Hexamers (639,546, Takara Bio, USA). Quantitative PCR was performed on reverse-transcribed cDNA using TaqMan™ Universal PCR Master mix (4,304,437, Applied Biosystem) to analyze mRNA expression levels. Relative expression of mRNA was determined by the ΔΔCt method.