Cell total rna isolation kit

The BMSCs were cultured in 12-well plates and exposed to 12.5 μg mL⁻¹ Ti₃C₂–PVP nanosheets and NIR light. Osteogenesis induction medium was prepared by supplementing the complete medium with 10 nM dexamethasone, 10 mM sodium β-glycerol phosphate disodium, and 50 μg mL⁻¹ ascorbic acid. After osteogenesis induction for 14 days, total RNA was extracted using Cell Total RNA Isolation Kit (Vazyme, China). Reverse transcription was performed to generate cDNA using HiScript III All-in-one RT SuperMix (Vazyme, China). Then, a 20 μL quantitative real-time PCR system was established using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China). Real-time fluorescence quantitative PCR analysis was performed using a real-time fluorescence quantitative PCR system (Bio-Rad, USA). Relative gene expression levels were determined by the ΔΔC_t method and normalized by GAPDH. Primer sequences used in the analysis are listed below:
GAPDH: F-5′-AGGTCGGTGTGAACGGATTTG-3′, R-5′-TGTAGACCATGTAGTTGAGGTCA-3′;
ALP: F-5′-TCCGTGGGCATTGTGACTAC-3′, R-5′-TGGTGGCATCTCGTTATCCG-3′;
OCN: F-5′-GGTAGTGAACAGACTCCGGC-3′, R-5′-GGCGGTCTTCAAGCCATACT-3′;
OPN: F-5′-ATCTCACCATTCGGATGAGTCT-3′, R-5′-TGTAGGGACGATTGGAGTGAAA-3′;
RUNX2: F-5′-GACTGTGGTTACCGTCATGGC-3′, R-5′-ACTTGGTTTTTCATAACAGCGGA-3′;
COL1: F-5′-GCTCCTCTTAGGGGCCACT-3′, R-5′-ATTGGGGACCCTTAGGCCAT-3′.

The total RNA of the cultured cells was extracted and purified by the Cell Total RNA Isolation Kit (Vazyme, RC112). The extracted RNA was reverse transcribed into cDNA using a Reverse Transcription Kit (Vazyme, R323). Real-time quantitative PCR (qPCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711). The total reaction system was 20 μL, including 0.5 μL cDNA obtained from reverse transcription of 1 μg RNA, 0.4 μL of forward and reverse primers (10 μM), 10 μL of 2 × ChamQ Universal SYBR qPCR Master Mix, and variable ddH₂O to generate a volume of 20 μL. The samples underwent two-step amplification with an initial step at 95°C (30 s), followed by 95°C (10 s) and 60°C (30 s) for 40 cycles. Fold changes in the expression of each gene were calculated by the relative quantitative method (2^-△△CT method).