Table 1.
PCR conditions for quantitative real-time PCR analysis of hamster and mouse models of TSE disease.
| Hamster | ||||||||
| Gene | Primer Pairs | Size (bp) | Denature 95°C (sec) | Anneal (°C) | Anneal Time (sec) | Extension 72°C (sec) | Mg2+ (mM) | Number Cycles |
| Mx2 | 5'-TGGCGGTAGGCATTCAGG-3' 5'-TGCCAGGACCAAGTTTACAGG-3' |
161 | 5 | 54 | 5 | 7 | 4 | 40 |
| 2'5'-OAS | 5'-AGCCGCTGCCCCCACTGTA-3' 5'-TCCGAGACTGCCCTGAAGC-3' |
109 | 5 | 57 | 5 | 5 | 3 | 50 |
| Eif2ak2 | 5'-TAGGCCTTGTCAACAGTTATGCTCA-3' 5'-GCTGCTTTGCCTCCTGCTTGGTAG-3' |
162 | 5 | 58 | 5 | 7 | 3 | 45 |
| GAPdH | 5'-ATGGCAAGTTCAAAGGCACAGTCA-3' 5'-TGGGGGCATCAGCAGAAGG-3' |
231 | 5 | 60 | 5 | 11 | 3 | 40 |
| GFAP | 5'-GGACATCGAGATTGCCACCTAT-3' 5'-CATCCCTCATCTCCACTGTCTTTA-3' |
176 | 5 | 60 | 5 | 8 | 3 | 45 |
| Mouse | ||||||||
| Gene | Primer Pairs | Size (bp) | Denature 95°C (sec) | Anneal (°C) | Anneal Time (sec) | Extension 72°C (sec) | Mg2+ (mM) | Number Cycles |
| Mx2 | 5'-CCTGCCTGCCATCGCTGTC-3' 5'-GCCTCTCCACTCCTCTCCCTCATT-3' |
160 | 5 | 60 | 5 | 11 | 3 | 40 |
| 2'5'-OAS | 5'-GAGGCGGTTGGCTGAAGAGG-3' 5'-GAGGAAGGCTGGCTGTGATTGG-3' |
312 | 5 | 60 | 5 | 11 | 3 | 40 |
| Eif2ak2 | 5'-GTACAAGCGCTGGCAGAACTCAAT-3' 5'-AAGAGGCACCGGGTTTTGTAT-3' |
124 | 5 | 60 | 5 | 11 | 4 | 40 |
| GAPdH | 5'-CACGGCAAATTCAACGGCACAGT-3' 5'-TGGGGGCATCGGCAGAAGG-3' |
232 | 5 | 60 | 5 | 11 | 3 | 40 |
| GFAP | 5'-GAGCGAGCGTGCAGAGATGATGG-3' 5'-CTCCCGAAGCTCCGCCTGGTAGA-3' |
166 | 5 | 60 | 5 | 11 | 3 | 45 |
| Caspase 8 | 5'-TCTGCTGGGAATGGCTACGGTGAA-3' 5'-GTGTGAAGGTGGGCTGTGGCATCT-3' |
212 | 5 | 60 | 5 | 11 | 3 | 45 |
| TNFα | 5'-TGCTCTGTGAAGGGAATGGGTGTT-3' 5'-AGTCCTTGATGGTGGTGCATGAGA-3' |
241 | 5 | 60 | 5 | 11 | 3 | 45 |
| TNFα Receptor | 5'-AACCAGTTCCAACGCTACCTGAGT-3' 5'-AAGGGACGCACTCACTTTCTCTCA-3' |
157 | 5 | 60 | 5 | 8 | 3 | 45 |
| Fas Ligand | 5'-ATCCCTCTGGAATGGGAAGACACA-3' 5'-ACCCAGTTTCGTTGATCACAAGGC-3' |
91 | 5 | 63 | 5 | 6 | 3 | 45 |
| Fas/ApoI Receptor | 5'-TCGCCTATGGTTGTTGACCATCCT-3' 5'-TGGTATGGTTTCACGACTGGAGGT-3' |
136 | 5 | 57 | 5 | 7 | 3 | 45 |
Conditions outlined above were used to determine the relative expression levels of the genes Eif2ak2, 2'5'-OAS, Mx2, GFAP, TNFα, TNFR1, FasL, Fas/ApoI, and caspase 8, as well as the reference gene GAPdH using the Roche LightCycler® 1.5.