2.4. RNA isolation and real-time PCR analysis
The differences between the messenger RNA (mRNA) levels of osteogenic, adipogenic and chondrogenic related genes in stem cell differentiation of andrographolide at different concentrations (1, 5, and 10 μM) were analyzed using real-time PCR. Total RNA was extracted on days 0 and 21 of each induction period using a FavorPrep Blood/ Cultured Cell Total RNA Mini Kit (FAVORGEN, Ping-Tung, Taiwan) according to the manufacturer’s instructions. Real-time PCR was conducted using SensiFAST™ SYBR® Lo-ROX one step kits. Quantitative mRNA analysis was performed by the Mx 3005p Real Time PCR system (Stratagene, Agilent Technologies Germany GmbH & Co. KG, Baden-Württemberg, Germany). Specific primer pairs for each gene as shown in Table 2. The expression level of each gene level was calculated by the comparative cycle threshold (CT) method and normalized with that of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A comparison of mRNA expression in each sample was calculated based on the differences in ΔCT of individual samples (ΔΔCT). Graphs show the relative expression levels compared with the control on day 0.