Nucleotide sequences of the primers used in RT-PCR and exon-intron PCR analysis subdivided in human dCK-specific primers, rat dCK-specific primers, and primers for housekeeping genes
| . | 5′-3′ primer sequence . | Annealings temperature . |
|---|---|---|
| Human dCK | ||
| Forward | ||
| A7-human | TCT TTG CCG GAC GAG CTC TG- | 65°C |
| T7-human | GGA TCC TAA TAC GAC TCA CTA | 55°C |
| TAG GAA CAG ACC ACC ATG GCC | ||
| ACC CCG CCC AAG AGA A– — | ||
| Exon 1 | CCG CCA GTG TCC TCA GCT GC- | 65°C |
| Exon 2 | CCT GAC AAC TTT TCT TCC TC- | 55°C |
| Exon 3 | CCT ATT GAC CAT TAA TTT GC- | 55°C |
| Exon 4 | GAT ACA TGT GTT GAT GAA GA- | 55°C |
| Exon 5-6 | CCC TGC CTT TTT CTT CCA TC- | 55°C |
| Exon 7 | GAT ACC TCA ATT AGT CAA GG- | 55°C |
| Reverse | ||
| B5-human | TGG AAC CAT TTG GCT GCC TG- | 65°C |
| B6-human | CAA GAT CAC AAA GTA CTC AA- | 55°C |
| Exon 1 | CTT GCG TCC CAC ATT TCC GG- | 65°C |
| Exon 2 | CAT ACC TCA AAT TCA TCT TG- | 55°C |
| Exon 3 | TTC AAA TGG CCA CGT ACA AG- | 55°C |
| Exon 4 | ATC CAC TGA AAA GAG TTC TA- | 55°C |
| Exons 5-6 | TTC TCA CCA CTC AGT ACA CT- | 55°C |
| Exon 7 | CAC CAT TAG GCT CAA TTC TA- | 55°C |
| Rat dCK | ||
| Forward | ||
| A7-rat | CCT GAG GTC CCG CGT CCT TA- | 65°C |
| T7-rat | GGA TCC TAA TAC GAC TCA CTA | 55°C |
| TAG GAA CAG ACC ACC ATG GCC | ||
| ACC CCA CCT AAG AGG — — | ||
| Reverse | ||
| B5-rat | TTG CCT GTT GTC TCC TGT GC- | 65°C |
| B6-rat | TGC AAT CAC AAA GTA CTC AA- | 55°C |
| Housekeeping genes | ||
| Forward | ||
| HPRT A | TGA CCA GTC AAC AGG GGA CA- | 55°C |
| GAPDH 5′ | ACC ACA GTC CAT GCC ATC AC- | 55°C |
| Reverse | ||
| HPRT B | CTT GAA CTC TGA TCT TAG GC- | 55°C |
| GAPDH 3′ | TCC ACC ACC CTG TTG CTG TA- | 55°C |
| . | 5′-3′ primer sequence . | Annealings temperature . |
|---|---|---|
| Human dCK | ||
| Forward | ||
| A7-human | TCT TTG CCG GAC GAG CTC TG- | 65°C |
| T7-human | GGA TCC TAA TAC GAC TCA CTA | 55°C |
| TAG GAA CAG ACC ACC ATG GCC | ||
| ACC CCG CCC AAG AGA A– — | ||
| Exon 1 | CCG CCA GTG TCC TCA GCT GC- | 65°C |
| Exon 2 | CCT GAC AAC TTT TCT TCC TC- | 55°C |
| Exon 3 | CCT ATT GAC CAT TAA TTT GC- | 55°C |
| Exon 4 | GAT ACA TGT GTT GAT GAA GA- | 55°C |
| Exon 5-6 | CCC TGC CTT TTT CTT CCA TC- | 55°C |
| Exon 7 | GAT ACC TCA ATT AGT CAA GG- | 55°C |
| Reverse | ||
| B5-human | TGG AAC CAT TTG GCT GCC TG- | 65°C |
| B6-human | CAA GAT CAC AAA GTA CTC AA- | 55°C |
| Exon 1 | CTT GCG TCC CAC ATT TCC GG- | 65°C |
| Exon 2 | CAT ACC TCA AAT TCA TCT TG- | 55°C |
| Exon 3 | TTC AAA TGG CCA CGT ACA AG- | 55°C |
| Exon 4 | ATC CAC TGA AAA GAG TTC TA- | 55°C |
| Exons 5-6 | TTC TCA CCA CTC AGT ACA CT- | 55°C |
| Exon 7 | CAC CAT TAG GCT CAA TTC TA- | 55°C |
| Rat dCK | ||
| Forward | ||
| A7-rat | CCT GAG GTC CCG CGT CCT TA- | 65°C |
| T7-rat | GGA TCC TAA TAC GAC TCA CTA | 55°C |
| TAG GAA CAG ACC ACC ATG GCC | ||
| ACC CCA CCT AAG AGG — — | ||
| Reverse | ||
| B5-rat | TTG CCT GTT GTC TCC TGT GC- | 65°C |
| B6-rat | TGC AAT CAC AAA GTA CTC AA- | 55°C |
| Housekeeping genes | ||
| Forward | ||
| HPRT A | TGA CCA GTC AAC AGG GGA CA- | 55°C |
| GAPDH 5′ | ACC ACA GTC CAT GCC ATC AC- | 55°C |
| Reverse | ||
| HPRT B | CTT GAA CTC TGA TCT TAG GC- | 55°C |
| GAPDH 3′ | TCC ACC ACC CTG TTG CTG TA- | 55°C |
The PCR reactions were performed in a reaction mixture containing 1.5-mmol/L MgCl2, 50-mmol/L KCl, 10-mmol/L Tris-HCl (pH 8.4), 0.2 mg of bovine serum albumin, 0.25-mmol/L of each deoxyribonucleoside triphosphate, 50 pmol of each primer, and 1 unit of Taq polymerase. The PCR was started after denaturation for 5 minutes at 95°C, followed by 30 to 33 cycles consisting of 48 seconds at 95°C, 48-seconds primer-specific annealing at 55 or 65°C and 48 seconds at 72°C, and a final elongation at 72°C for 5 to 10 minutes.