Quantinova syber green pcr kit

The osteosarcoma cells were first lysed with TRIzol (9,109; TaKaRa, Tokyo, Japan), then whole RNA was extracted using standard chloroform/isopropanol method. The RNA samples were quantified by using NanoDrop, and cDNA was synthesized by using reverse transcriptase enzyme (205,311; Qiagen, Duesseldorf, Germany). Relative expression of genes was studied by performing real-time polymerase chain reaction (PCR) using Quantinova Syber green PCR kit (208,054; Qiagen). GAPDH was used as internal control. Primer sequences are listed in the Supplementary Table S1.

For validation of mRNA and miRNA sequencing expression results, 11 DEGs (including 6 hub genes) and 7 DEmiRNAs were randomly selected and analyzed by RT-qPCR. Primers were designed using Primer 5.0 software (Supplementary Table S1) and synthesized by Sangon Biotech (Shanghai) Co. Ltd. RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States) was used to reverse transcribe the total RNAs into cDNA following the manufacturer’s protocols for mRNA. Then qPCR was conducted using QuantiNova SYBR Green PCR Kit (QIAGEN, Shanghai, China). For miRNA, specific reverse transcription primers with step loop were synthesized and reverse transcription were performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States). The qPCR was completed using QuantiNova SYBER Green PCR Kit (QIAGEN, Shanghai, China). GAPDH gene and U6 were used for normalizing the relative abundance of genes and miRNAs, respectively. The 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used to analyze the data for all samples in triplicate technical replicates.