Real-Time PCR Housekeeping Gene Analysis

β-ACTIN and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were selected as housekeeping genes based on previous analyses from our laboratory that demonstrated high stability in the bfMSC cultures (4 (link), 6 (link)). Primers were designed using the PrimerExpress software (Applied Biosystems Incorporated, Foster City, CA, USA) (Table 1). Equivalence of amplification efficiencies among all primer-probe sets was confirmed using serial 3-fold dilutions of AT-MSC cDNA. Each PCR reaction (10 μL) contained the following: 2X Brilliant II SYBR Green QPCR master mix (5 μL; Agilent Technologies), target forward primer (200 nM), target reverse primer (200 nM), cDNA synthesis reaction (1 μL), and nuclease-free PCR-grade water to adjust the final volume. The PCR amplification was carried out in an Eco Real-Time PCR System (Illumina Incorporated, San Diego, CA, USA). Thermal cycling conditions were 95°C for 10 min, followed by 40 repetitive cycles at 95°C for 30 s, and 60°C for 1 min. All reactions were performed in triplicate. In each experiment, the amount of gene expression was recorded as CT values that corresponded to the number of cycles where the fluorescence signal can be detected above a threshold value. The CT averages for each biological replicate were calculated and transformed into relative values through the ΔΔCT formula (24 (link)).