Qrt pcr

The cells were cultured as described in Section 2.5. Total cellular RNA in each well was collected using Total RNA Kit I (Omega R6834-01, Omega, Guangzhou, China) following the manufacturer’s instructions and the concentration of acquired RNA was measured using a NanoDrop^TM 2000 spectrophotometer (ThermoFisher, Waltham, MA, USA). The RNA from each group was then reverse transcribed into complementary DNA (cDNA) using TransScript II All-in-one Fist-Strand cDNA Synthesis SuperMix. The cDNA was then amplified and analyzed by qRT-PCR (TransGen Biotech, Beijing, China) with TransStart Green qPCR SuperMix (TransGen Bioteh, Beijing, China) and primers. The relative expressions of osteoblastic differentiation-related genes, including ALP, osteopontin (OPN), RUNX family transcription factor 2 (RUNX2), collagen-I (COL-I), and osteocalcin (OCN) were quantified using the cycle threshold value and 2^−ΔΔCT method. The expression of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for normalization. PCR primers sequences are provided in the Table S1.

200 ng total RNA was isolated from plant samples using the RNAprep Pure Plant Plus Kit (TransGen Biotech Co., Ltd., Beijing, China). The extracted RNA was measured by spectrophotometer 260/280, and RNAs with OD value between 1.8–2.0 were selected for reverse transcription. 200 ng of total RNA was reverse-transcribed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd., Beijing, China) in a final volume of 100 µL, Table 2 is the procedure of real-time PCR. All primers were designed by Primer5 software and synthesized commercially (Sangon Biotech Co., Ltd., Shanghai, China) (Table S1). The PpActin gene served as an internal control to normalize the expression levels [23 (link)]. qRT–PCR (TransGen Biotech Co., Ltd., Beijing, China) was performed using the LightCycler^®96 quantitative PCR platform. To be sure of the absence of primer dimers or non-specific products, all samples we performed a Melting curve analysis from a real time PCR assay, and the experiments were independently repeated three times. Then relative expression levels were measured using the 2−ΔΔCt method [53 (link)]. The variance analysis was shown in Tables S4 and S5.