Superprep cell lysis rt kit for qpcr

A549 cells were treated with trypsin, suspended in fresh medium, and seeded onto 96-well culture plate at a cell number of 1 × 10⁴ cells/well. After overnight incubation, culture medium was replaced with reduced serum DMEM containing 0.1% FBS. The day after medium replacement, cells were treated with premixtures of TGF-β1 (PeproTech) and each aptamer or anti-pan-TGF-β antibody (R&D Systems) at indicated concentrations for 24 h, which were diluted with DMEM without serum. The treated cells were washed with phosphate-buffered saline (PBS), lysed, and then subjected to cDNA synthesis using a SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo, Osaka, Japan) according to manufacturer’s protocol. The synthetic cDNAs were examined by real-time PCR (qPCR) using an AB 7300 Real-Time PCR System (Applied Biosystems) and a TaqMan Universal PCR Master Mix. TaqMan probes for human COL1A1, VIM, and CDH1 were purchased from Life Technologies.

RAW264.7 cells were cultured in 96-well plates at a density of 1.0 × 10⁴ cells/well, and each extract and LPS were added after 24 h of culture. After another 24 h of culture, cells were collected, and cDNA was prepared using the SuperPrep^® Cell Lysis & RT Kit for qPCR (TOYOBO, Osaka, Japan). RT-qPCR was performed using THUNDERBIRD^® Next SYBR^® qPCR Mix (TOYOBO) and QuantStudio 5 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA), and the relative expression levels between samples were evaluated using the ΔΔCt method. Primer information is shown in Table 1.

Huh-7 cells, a human hepatoma cell line, was cultured at 37 °C, 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. Huh-7 cells were seeded at 10,000 cells/well in 9 mM Ca²⁺ enrichment medium (CEM) containing ASOs or BRO at a final concentration of 1 µM in 96-well plates. After 24 h, cells were washed with PBS, and cDNA was synthesized directly from cell lysates using a SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo). qRT-PCR was performed using Fast SYBR® Green Master Mix (Thermo Fisher Scientific) and analyzed using the StepOnePlus system (Applied Biosystems).

Total RNA was isolated and then reverse‐transcribed by using the SuperPrep® Cell Lysis & RT Kit for qPCR (SCQ‐101; Toyobo Co., Ltd.) on day 8. Quantitative RT‐PCR for muscle‐related genes was performed with SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (#RR820Q; Takara Bio Inc.,) according to the manufacturer's instructions. Primer information can be found in Table S2.

At 48 h post transfection, cell lysis and reverse transcription were performed using a SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo, Osaka, Japan), in accordance with the manufacturer's instructions. Relative mRNA expression levels were quantified by quantitative real-time PCR (qRT-PCR) using a KOD SYBR qPCR Mix (Toyobo) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Expression levels of CDH1 and RANKL were normalized by that of RPL8. Relative expression changes were calculated using the relative standard curve method. The primers used are listed in the Supplementary Sequences.

For the in vitro mRNA silencing assay, CD206-expressing cells were seeded in 96-well plates. Cells were then treated with appropriate compounds for 4 days, and cDNA was prepared with a SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo, Osaka, Japan) according to the manufacturer's protocol. The transcript levels of HPRT1 and ACTB were measured using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) and QuantStudio 12K flex (Thermo Fisher Scientific). TaqMan primer sets for human HPRT1 (code no. Hs99999909_m1) and human ACTB (code no. Hs01060665_g1) were purchased from Thermo Fisher Scientific. The relative mRNA expression was quantified using the comparative Ct method. For the B2M gene, TaqMan primer sets for human B2M (code no. Hs00984230_m1) and human GAPDH (code no. Hs02786624_g1) were used. For in vivo evaluations, splenic and hepatic F4/80-positive cells were lysed using TRIzol Reagent (Thermo Fisher Scientific). The total RNA was then collected with a RNeasy Mini Kit (Qiagen) and converted to cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche Life Science). qPCR was conducted as described above using TaqMan primer sets for mouse Hprt1 (code no. Mm01545399_m1), mouse B2M (code no. Mm00437762_m1), and mouse Actb (code no. Mm00607939_s1).