Hiscript 3 rt supermix for qpcr

To extract RNA from tissues, tissues were homogenized in TRIzol reagent (Vazyme, Nanjing, China) with beads and were then placed on ice for 30 min. After that, ice-cold chloroform, isopropanol, and 75% ethanol were used sequentially for RNA extraction. To extract RNA from cultured cells, the same protocol was followed, except that the step of homogenization with beads was omitted. Reverse transcription was performed with HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China) and the expression levels of targeted genes were evaluated via qPCR with ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China). β-actin was used as the internal reference gene. The primer sequences are listed in Supplementary Table 1.

Total RNA was extracted from the cortex of rats and HEK293 cells using the Ultrapure RNA Kit (CWBIO, Beijing, China). miRNA 1st Strand cDNA Synthesis Kit and miRNA Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) were used to measure the expression of DEMs in the 2VO rat model. Likewise, the DEGs in the rats with 2VO were also measured using Hiscript^®III RT SuperMix for qPCR and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses for miRNA and mRNA were performed following the manufacturer’s recommendations. The expression of miRNA and mRNA was calculated by the 2^–ΔΔCT method (Yi et al., 2021 (link)). The primes in this study were synthesized from Sangon Biotech (Shanghai, China) and are listed in Table 1. U6 small RNA was used as the internal reference for miRNA. Gapdh and ACTB were used as the reference genes for mRNA.

TRIzol reagent (Genstar, Cat#P118-05) and chloroform were added to homogenize single cells, followed by RNA precipitation, washing and resuspension following the manufacturer’s protocol. The extracted RNA was used for reverse transcription according to the manufacturer’s protocol (HiScript III RT SuperMix for qPCR, Vazyme, R323-01). Quantitative RT-PCR analysis was performed with SYBR Select Master Mix (Genstar, Cat#A301-10) using StepOne Plus (Life Sciences). All data were normalized to β-Actin expression. qPCR primer sequences are listed in Supplementary Table 1.

cDNAs were generated from the RNA using HiScript III RT SuperMix for qPCR (Vazyme), which includes Random primers/Oligo(dT)20VN primer mix for reverse transcription. qPCR was performed using a MyIQ2 real-time PCR system (Bio-Rad) with AceQ SYBR Green Master mix (Vazyme). The primers used in qRT-PCR are listed in Supplementary Table 11.

Total RNA from cells were isolated by Trizol (Takara Biotechnology (Dalian), Dalian, Chian), which was then used as the template for reverse transcription. HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China) was utilized to synthesize cDNA in accordance with the manufacturer’s instructions. Utilizing AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), qPCR was performed. The system (10 µL) contained 1 µL template of cDNA, 5 µL of 2 × AceQ Universal SYBR qPCR Master Mix, 0.2 µL of each primer and 3.6 µL of deionized water. The following were the reaction conditions: 95 ℃ for 5 m, 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. The level of mRNA expression was normalized with GAPDH. The sequence of primers used in the qPCR assays are shown in Table S1.

To identify the osteoinductive activity of factors released from rBMP-2/SF microspheres, P3 ADSCs from OI-modeled mice were co-cultured with 20 µL of blank SF or rBMP-2/SF microspheres for 7 days. The medium was replaced on the third day and supplemented with an equivalent dosage of microspheres. Defective and wild-type ADSCs under normal culture served as control cells. ADSCs^WT under normal culture conditions acted as the control. The extraction of total RNA from ADSCs was performed utilizing Trizol reagent (Invitrogen, USA) following the manufacturer’s instructions. Reverse Transcription of mRNA to cDNA was performed using HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China). Real-time qPCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme). The procedures were established using the protocols reported in a prior report (Liu et al., 2019 (link)). The mRNA expression levels of several osteogenesis-related genes were normalized to Glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The primers given in Table 1 were produced by Sangon Biotech Co., Ltd (Shanghai, China).