2.2.3. Quantitative RT-PCR of MSCs in microniches

Total RNA of MSCs in microniches was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For each sample, reverse transcription was performed using the ReverTra Ace Qrna RT Master Mix with gDNA Remover (TOYOBO). Quantitative RT-PCR (qRT-PCR) was performed using the CFX96 Real-Time System (Bio-Rad, USA). Samples were denatured for 30 s at 95 °C, and then amplified for 40 cycles as follows: denaturation at 95 °C for 5 s, annealing at 55 °C for 10 s, and extension at 72 °C for 15 s. The Ct values of the products were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, and the expression levels of the genes of interest were calculated using the 2^−ΔΔCT method. Primer details are shown in Table 1.

Details of primer.