Table 2.
Primers used for Real time qPCR (RT) and probes for in situ hybridization (ISH)
| Gene | Orientation | Genbank | Tm | Use | Sequence (5'-3') |
|---|---|---|---|---|---|
| Extracellular Matrix constituents | |||||
| Col1a1 | Forward | FJ195608 | 68.6 | RT | AGAGAGGAGTCATGGGACCCGT |
| Reverse | 68.8 | RT | GGGTCCTGGAAGTCCCTGGAAT | ||
| Forward | 69.8 | ISH | TAGCCGTGGTTTCCCTGGTT | ||
| Reverse | 68.7 | ISH | CCGGGAGGTCCAAATCTACC | ||
| Col2a1 | Forward | FJ195613 | 62.6 | RT | TGGTCGTTCTGGAGAGACT |
| Reverse | 62.7 | RT | CCTCATGTACCTCAAGGGAT | ||
| Forward | 64.4 | ISH | GCTGGCGAGACAGGAGAGA | ||
| Reverse | 63.4 | ISH | GCCTCATCAGCCCTCATGTA | ||
| Col10a1 | Forward | EG837148 | 66.4 | RT | TGGTGCTCTTTGACTGCCTGTAA |
| Reverse | 65.1 | RT | CATCCTGTGTGTTGCAATATCACA | ||
| Forward | 61.9 | ISH | AACAAGGGCTTCTTGGATCA | ||
| Reverse | 60.9 | ISH | CATAATGCATCCTCAGGCAT | ||
| ALP | Forward | FJ195609 | 62.6 | RT | CTAGTTTGGGTCGTGGTATGT |
| Reverse | 62.2 | RT | TGAGGGCATTCTTCAAAGTA | ||
| Osteocalcin | Forward | FJ195611 | 62.9 | RT | GTGAACCAACAGCAAAGAGA |
| Reverse | 62.8 | RT | CCAGGTCCTTCTTAACAAACA | ||
| Forward | 59.6 | ISH | CTCATACTTGTTGATCGTCCAG | ||
| Reverse | 60.2 | ISH | TCTTTCTCTCTCGCTCTCCC | ||
| Osteonectin | Forward | FJ195614 | 65.3 | RT | ATTACTGAGGAGGAGCCCATCATT |
| Reverse | 65.9 | RT | CCTCATCCACCTCACACACCTT | ||
| Forward | 64.6 | ISH | CTGAACGATGAGGGTGTGGA | ||
| Reverse | 67.2 | ISH | CGAGTGGTGCAGTGCTCCAT | ||
| Decorin | Forward | DQ452069 | 65.0 | RT | GAACCTGGCTAAGCTGGGTCTAA |
| Reverse | 66.0 | RT | GAACAGGCTGATGCCAGAGTACAT | ||
| MMP9 | Forward | CA342769 | 65.9 | RT | AGTCTACGGTAGCAGCAATGAAGGC |
| Reverse | 65.7 | RT | CGTCAAAGGTCTGGTAGGAGCGTAT | ||
| MMP13 | Forward | DW539943 | 66.9 | RT | TGATGTCCAAGTCAGCCGCTTC |
| Reverse | 63.6 | RT | AAGGAGGCAGGAGGAAGAGG | ||
| Vimentin | Forward | GE618371 | 62.5 | RT | CAAGATCCTGTTAGCAGAGCT |
| Reverse | 67.8 | RT | TGGTCTGCCACTTGCGATTGTC | ||
| Transcription factors | |||||
| Sox9 | Forward | EU344852 | 62.8 | RT | CCTGCAAACAAGACAAGGT |
| Reverse | 62.8 | RT | GGGTCGAGTAGATTCATACGA | ||
| Runx2 | Forward | FJ195615 | 64.8 | RT | CCACCAGGGACAGACACAGAT |
| Reverse | 64.0 | RT | GAACGGACTGAGATCTGACGAA | ||
| Osterix | Forward | FJ195612 | 63.0 | RT | TCCCATAGACTTTCCCACA |
| Reverse | 62.9 | RT | TGCCTCAGGACATGTACAA | ||
| Mef2c | Forward | GU252207 | 62.5 | RT | CACCGTAACTCGCCTGGTCT |
| Reverse | 62.3 | RT | GCTTGCGGTTGCTGTTCATA | ||
| Twist* | Forward | AY546100 | 61.8 | RT | GCTTCAAAAGTGGAGACCGTTT |
| Reverse | 61.6 | RT | GGGAGAACTTGAGCCCTCTTC | ||
| Signalling molecules: | |||||
| BMP4 | Forward | FJ195610 | 62.2 | RT | TCAAGTTGCCCATAGTCAGT |
| Reverse | 62.6 | RT | CACCTGAACTCTACCAACCA | ||
| BMP2 | Forward | BT059611 | 62.5 | RT | ATGTGGTATTGCACCCATT |
| Reverse | 62.9 | RT | ATGGACAGTTTCCCAATGA | ||
| Shh* | Forward | AY370830 | 61.7 | RT | CCGGCTCATGACTCAGAGATG |
| Reverse | 62.2 | RT | TATCCCTGGCCACTGGTTCA | ||
| Ihh | Forward | FJ195617 | 63.2 | RT | GGCTCAATCTCCTCTCTCCAAT |
| Reverse | 63.3 | RT | GCTTGGTTGGGAGATATGCA | ||
| PDGFrb | Forward | BT071824 | 62.2 | RT | TAGGACCAGCCGATGTTACTGC |
| Reverse | 62.9 | RT | TCTCTGAGCCTCTCGATGT | ||
| Housekeeping genes: | |||||
| EF1a** | Forward | DQ834870 | 71.3 | RT | CACCACCGGCCATCTGATCTACAA |
| Reverse | 69.4 | RT | TCAGCAGCCTCCTTCTCGAACTTC | ||
| GAPDH*** | Forward | BT043825.1 | 72.4 | RT | TGGTGCAGAACCTCATGGTCCTCA |
| Reverse | 70.2 | RT | ATCCCGGATGATTCCAAAGTCGTC | ||
| Hsp90β *** | Forward | BX074486 | 59.7 | RT | AATGGGTAACCTGGTCAGTG |
| Reverse | 60.6 | RT | GACTCAGGCAAAGGAACCTT | ||