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