Rnafast200

Total RNA was extracted from cells or tissues using RNAfast200 (Fastagen, Shanghai, China) or Trizol (TIANGEN BIOTECH, Beijing, China), and reversely transcripted into cDNA with ReverTra Ace qPCR RT Kit (TOYOBO Life Science, Shanghai, China). qPCR was carried out using SYBR Green Master Mix (CWbiotech, Beijing, China). The relative mRNA levels of interested genes were evaluated using 2^-△△Ct method, using 18 s rRNA or GAPDH as internal control. The primers were listed (Additional file 1: Table S2).

Total cellular RNA was isolated using the RNA Fast 200 (Cat. #220010; Fastagen Biotech, Shanghai, China) and reversely transcribed into cDNA using the PrimeScript™ RT Master Mix (cat. #RR036A, TaKaRa Biotechnology (Dalian) Co., Ltd., China) according to the manufacturers’ protocols. PCR was performed using the TB Green™ Premix Ex Taq™ II (Cat. #RR820A, TaKaRa) in Bio-Rad CFX96 system (Hercules, CA, USA) according to the manufacturer’s instructions. The primers used were PATJ, 5′-AAG GGT GAC ACG TCG CAG AA-3′ and 5′-GGC TGA ACA ATC TGA GGG TAT ATG G-3′; β-actin, 5′-CAT GTA CGT TGC TAT CCA GGC-3′ and 5′-CTC CTT AAT GTC ACG CAC GAT-3′. The experiment was performed in triplicate and level of PATJ mRNA was normalized to β-actin.

Human CDK3 siRNA (siCDK3) and nonspecific control siRNA (siNC) were synthesized and provided by JTSBio. According to the manufacturer's protocol, OS cells were seeded in a six‐well plate at 2.5 × 10⁵ cells per well and transfected with jetPRIME transfection reagent (Polyplus, France). We extracted the total RNA of these cells with Rnafast200 (Fastagen, Japan) 48 h after transfection. HiScript II Q RT SuperMix (Vazyme, China) synthesized cDNA for RT‐qPCR. The PCR amplification system was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) based on the manufacturer's introduction. The PCR reaction was performed according to the following steps: initial denaturation at 95°C for 30 s, 1 repetition; denaturation at 95°C for 10 s, 60°C for 30 s, 40 repetitions; dissolution curve at 95°C for 15 s, 60°C for 60s, 95°C for 15 s, 1 repetition. Gene expression was normalized based on GAPDH and calculated by the lg2–△△Ct method. The sequences of siRNA and primers are listed in Table S3.

Total mRNA was extracted using RNAfast200 (Fastagen, China) and then converted into cDNA using a reverse transcription kit (TOYOBO, Cat. FSQ-101), according to the manufacturer’s instructions. In order to examine the levels of gene expression, the qRT-PCR analysis provided by the Kit (TAKARA, Cat. RR820A). Internal controls in the form of GAPDH and actin mRNA as housekeeping genes were employed. Table 1 contains the list of primers utilized for the target genes.