Sybr1 premix ex taqtm

MC3T3-E1 cells were grown in a differentiation-inducing medium for 24 hours, and total RNA was isolated using Beyozol reagent (Beyotime). The RNA integrity was determined by separating the RNA on an agarose gel and quality was assessed by measuring the A260/A280 and A260/A230 ratio cutoff higher than 1.8 and 2.0, respectively. Two micrograms of total RNA were used for reverse transcription to synthesize cDNA with dNTP mix and RNase-free H₂O, following the manufacturer's protocols. qRT-PCR was performed using SYBR1 Premix Ex Taq^TM (Takara, Dalian, Japan). The qRT-PCR parameters were as follows: 95.0°C for 30 s, 39 cycles of 95.0°C for 5 s and 60.0°C for 30 s, and 95.0°C for 10 s, followed by a melt curve from 65.0 to 95.0°C at an increment of 0.5°C per 5 s. Three samples were obtained from three independent experiments, and each sample was analyzed in triplicate. The gene expression data were normalized to an internal control (GAPDH) and were analyzed using the comparative 2^-ΔΔCq method only when amplification efficiency calculated using the standard curve of the mRNAs of interest and the internal mRNA was 90%~110% 32 (link). GraphPad Prism 7 was used to calculate the data as fold changes compared with the control group. The primer sequences used are shown in Table 1.

The total RNA of cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 μg of total RNA by incubating for 1 h at 42°C with Superscript III reverse transcriptase (Invitrogen, Mulgrave, Australia) following oligo(dT) priming. After reverse transcription reaction, qRT-PCR was performed by LightCycler480 system (Roche, Mannheim, Germany) using SYBR1Premix Ex TaqTM (Takara, Dalian, China) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Data were analyzed using the comparison Ct (2^-ΔΔCt) method [51 (link)] and expressed as fold change compared to respective control. Each sample was analyzed in triplicate. The primer sequences used in this study were as follows:
GAPDH: forward,5′-GACTTCAACAGCAACTCC CAC-3′; reverse, 5′-TCCACCACCCTGTTGCTGTA-3′; Collagen I (Col I): forward, 5′-GAGCTGGTGTAA TGGGTCCT-3′; reverse, 5′-G AGACCCAGGAAGACC TCTG-3′; bsp: forward, 5′-AGTGTGGAAAGTGTGG CGT T3′; Ocn: forward, 5′-AAGCAGGAGGGCAATA AGGT-3′; reverse, 5′-TTTGTAGGC GGTCTTCAAGC-3′; and SATB2: forward, 5′-GCCGTGGGAGGTTTGATG ATT-3’; reverse, 5′-ACCAAGACGAACTCA- GCGTG-3′.