Quantitative real-time PCR analysis
The level of miR-195-5p after transfection and mRNA expressions of
osteogenic-related genes runt-related transcription factor 2 (Runx2) and
osteopontin (OPN) during osteogenic differentiation were evaluated by
real-time quantitative reverse transcription PCR (RT-qPCR) as previously
described (Li et al. , 2018). The primer sequences used were as
follows: Runx2, forward 5’-CAGACACAATCCTCCCCACC-3’, and reverse
5’-GCCAGAGGCAGAAGTCAGAG-3’; OPN, forward 5’-TCAAGGTCATCCCAGTTGCC-3’, and
reverse 5’-GACTCATGGCTGGTCTTCCC-3’; β-actin, forward
5’-CTCTGTGTGGATTGGTGGCT-3’, and reverse 5’-CGCAGCTCAGTAACAGTCCG-3’;
miRNA-195-5p, forward 5’- CGTTATCCTAGCAGCACAGAAAT-3’, and reverse
5’-TATGGTTTTGACGACTGTGTGAT-3’; and U6, forward
5’-CAGCACATATACTAAAATTGGAACG-3’, and reverse 5’-ACGAATTTGCGTGTCATCC-3’.
Each sample was tested in triplicate and the relative gene expressions
were calculated using the 2-ΔΔCT method, with β-actin
or U6 for normalization.