Sequences, probe and hybridisation information and references of AP-conjugated cDNA probes used for in situ hybridisation
| . | . | . | . | . | Hybridisation conditions . | . | . | . | |
|---|---|---|---|---|---|---|---|---|---|
| Target mRNA . | Probe sequence (5′-3′) . | No. of bases . | % GC . | Base no. of cDNA . | SSC . | % F . | Tm (°C) . | Reference . | |
| α1a | CCAAAGGCAAATGGGTTTAATATCATTG | 28 | 35.7 | 3197-3224, inside cds. | 4× | 35 | 55.8 | O. mykiss/AY319391 | |
| α1b | GCAGCTGAGTGCACCATCACAGTGTTCA | 28 | 53.6 | 3345-3372, inside cds. | 4× | 50 | 58.1 | O. mykiss/AY319390 | |
| α1c | CGGGGTAACACCTCAGAGAACTTGGTCC | 28 | 57.1 | 3409-3436, inside cds. | 4× | 50 | 60.5 | O. mykiss/AY319389 | |
| α3 | GACAGAAAGGGGGACCGGGGACGCCATG | 28 | 67.9 | 3872-3900, outside cds. | 1.5× | 50 | 60.6 | O. mykiss/AY319388 | |
| β-Actin | CCAGACTCGTCGTACTCCTGCTTGCTGA | 28 | 57.1 | 1070-1097, inside cds. | 4× | 50 | 60.5 | S. salar/AF012125 | |
| . | . | . | . | . | Hybridisation conditions . | . | . | . | |
|---|---|---|---|---|---|---|---|---|---|
| Target mRNA . | Probe sequence (5′-3′) . | No. of bases . | % GC . | Base no. of cDNA . | SSC . | % F . | Tm (°C) . | Reference . | |
| α1a | CCAAAGGCAAATGGGTTTAATATCATTG | 28 | 35.7 | 3197-3224, inside cds. | 4× | 35 | 55.8 | O. mykiss/AY319391 | |
| α1b | GCAGCTGAGTGCACCATCACAGTGTTCA | 28 | 53.6 | 3345-3372, inside cds. | 4× | 50 | 58.1 | O. mykiss/AY319390 | |
| α1c | CGGGGTAACACCTCAGAGAACTTGGTCC | 28 | 57.1 | 3409-3436, inside cds. | 4× | 50 | 60.5 | O. mykiss/AY319389 | |
| α3 | GACAGAAAGGGGGACCGGGGACGCCATG | 28 | 67.9 | 3872-3900, outside cds. | 1.5× | 50 | 60.6 | O. mykiss/AY319388 | |
| β-Actin | CCAGACTCGTCGTACTCCTGCTTGCTGA | 28 | 57.1 | 1070-1097, inside cds. | 4× | 50 | 60.5 | S. salar/AF012125 | |
Probes were labelled with alkaline phosphatase at the 5′-terminus. cds, coding sequence; SSC, saline sodium citrate; % F, percent formamide;Tm, theoretical melting of DNA–RNA duplexes point using listed hybridisation conditions calculated according to Wilkinson, 1992 as Tm = 79.8 + 18.5 log (molarity of monovalent cations) + 0.58 (% GC content of probe) + 0.0012 (% GC)2 – 0.5 (% formamide)– 820 / (probe length in bases)