Quantitative Analysis of Embryonic and Postnatal Gene Expression

Total RNA was isolated from pups of E18.5 and P0 using the TRIzol reagent (Invitrogen) as previously described.^{63 (link)} Reverse transcription was carried out utilizing the Evo M-MLVRT Premix (Applied Biosystems, Foster City, CA, USA). Quantitative RT–PCR was conducted with SYBR Green Pro Taq HS (Accurate Biotechnology (human) Co., Ltd) and the 7500 Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA). The primer sequences are as follows: Gapdh, 5’-CCACTCTTCCACCTTCG-3’ and 5’-GTGGTCCAGGGTTTCTTAC-3’; Col1a1, 5′-TAGGCCATTGTGTATGCAGC-3′ and 5′-ACATGTTCAGCTTTGTGGACC-3′; Osx, 5′-ATGGCGTCCTCTCTGCTTG-3′ and 5′-TGAAAGGTCAG CGTATGGCTT-3′; Runx2, 5′-TCCACSSGGACAGAGTCAGATTACAG-3′ and 5′-CAGAAGTCAGAGGTGGCAGTGTCATC-3′; Alp, 5′-CGGGACTGGTACTCGGATAA-3′ and 5′-ATTCCACGTCGGTTCTGTTC-3′; Pck2, 5′-CCCTGACTGGACATGGGGAT-3′, and 5′-GGCAAAGCACTTCTTGCCCA-3′.