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.