Rt pcr premix kit

The RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) was used to extract total RNA. Total RNA was then quantified using a spectrophotometer at 260 nm. A Thermal cycler (C1000 Thermal Cycler; Bio-Rad, Hercules, CA, USA) was used to make cDNA from the total RNA at 42 °C for 1 h and 95 °C for 5 min using a first-strand cDNA synthesis kit (Bioneer, Daejeon, Korea) and oligo (dT) primers. MMP2, MMP3, MMP9, VEGF, NANOG, SOX2, OCT4, and GAPDH cDNA were amplified using the RT-PCR Premix Kit (Bioneer) with primers synthesized by Bioneer. A LightCycler 480II (Roche) was used for qPCR as follows: 2 μL diluted cDNA was mixed with 1 μL forward and 1 μL reverse primers and 10 μL TB Green Advantage Premix (Takara Bio, Japan). The cycling conditions were as follows: 5 min for initial denaturation at 95 °C, followed by 40 cycles of 40 s at 95 °C, 40 s at 58 °C, 40 s at 72 °C, and 5 min at 72 °C. All reactions were performed in triplicate and normalized to GAPDH. The primers are listed in Supplementary Table S1. The calculations were carried out using the Cp values.

Alveolar bones and gingival tissues of rat first molars were collected from half number of each group at each time point (C, n = 3; P, n = 3; DP, n = 4; DP+IFX, n = 4 at each time point). Total RNA was extracted from alveolar bones and gingival tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. One μg of total RNA was converted to complementary DNA (cDNA) using RT-PCR PreMix Kit (Bioneer, Seoul, Korea) according to the manufacturer’s instructions. Quantitative real-time PCR was conducted using SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and a MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Specific real-time PCR primers for IL-1β, sclerostin, and GAPDH were listed in S1 Table. Relative gene expression quantification was normalized to GAPDH mRNA expression using 2^-∆∆ Ct analysis method. We performed melting curve analyses to verify amplification specificity and the absence of primer dimers.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). The RNA was quantified spectrophotometrically at 260 nm, and cDNA was synthesized from the total RNA at 42 °C for 1 h and at 95 °C for 5 min using a first-strand cDNA synthesis kit (Bioneer Corporation, Daejeon, Korea) and oligo d(T) primers. The RT-PCR Premix Kit (Bioneer Corporation) was used to amplify CCND1, CCNE1, CDK4, CDKN1A, CDKN1B, BAX, BCL-2, CYCS, CASP9, and GAPDH cDNA with primers synthesized by the Bioneer Corporation. qPCR was conducted in a thermal cycler (C1000 Thermal Cycler, Bio-Rad, Hercules, CA, USA) as follows: 2 μL of diluted cDNA was mixed with forward and reverse primers (1 μL each, 100 pM) and 10 μL of TB Green Advantage Premix (Takara Bio, Shiga, Japan). qPCR conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 40 s, annealing at 58 °C for 40 s, extension at 72 °C for 40 s, and a final extension at 72 °C for 5 min. The primers used for the amplification are listed in Table S1. All measurements were performed in triplicate. The relative expression of the target genes was normalized to GAPDH. The calculations were carried out using the Cp values.