Table 1.
Real-time PCR | |||||
---|---|---|---|---|---|
Ligands | Probes | Conditions |
|||
EGF | Hs00153181_M1 (Applied Biosystem) | 50°C 2′–95°C 10′–95°C 15′, 60°C 1′ × 40 cycles | |||
TGFα | Hs00608187_M1 (Applied Biosystem) | ||||
PTEN | Hs02621230_S1 (Applied Biosystem) | ||||
S6 | Hs01058685_g1 (Applied Biosystem) | ||||
Molecular analysis | |||||
Gene | Exon | Sense | Antisense | Annealing temperature (°C) | |
EGFR | 18 | ctgaggtgacccttgtctc | tacagcttgcaaggactctg | 60 × 40 cycles | |
19 | agatcactgggcagcatgtg | cagctgccagacatgagaa | |||
20 | cttctggccaccatgcgaa | ccaggcaaactctgctatcc | |||
21 | tcttcccatgatgatctgt | cctggtgtcaggaaaatgct | |||
PI3K | 9 | gggaaaaatatgacaaagaaagc | ctgagatcagccaaattcagtt | 58 × 35 cycles | |
20 | ctcaatgatgcttggctctg | tggaatccagagtgagctttc | |||
PTEN | 5 | tgcaacatttctaaagttacctact | gaggaaaggaaaaacatcaaaaa | 55 × 40 cycles | |
6 | tttttcaatttggcttctcttttt | tgttccaatacatggaaggatg | |||
7 | cagttaaaggcatttcctgtg | ttttggatatttctcccaatgaa | |||
8 | tgtcatttcatttctttttcttttc | aagtcaacaacccccacaaa | |||
9 | tcatggtgttttatccctcttg | tgagtcatatttgtgggtttca | |||
K-Ras | 1 | tggtggagtatttgatagtgt | catgaaaatggtcagaga | 56 × 35 cycles | |
2 | ggtgcactgtaataatccagac | tgatttagtattatttatggc | 50 × 45 cycles | ||
B-Raf | 11 | tccctctcaggcataaggtaa | cgaacactgaatatttcctttgat | 55 × 40 cycles | |
15 | tcataatgcttgctctgatagga | ggccaaaaatttaatcagtgga | 56 × 40 cycles | ||
Immunohistochemical analysis | |||||
Antibody | Clone | Company | Dilution | Antigen retrieval | Positive control |
PDGFRB | sc-339 | Santa Cruz Biotecnology | 1:100 | 6′ at 95°C mM citrate buffer, pH 6 | Human breast carcinoma |
EGFR | K1492 | Dako | Procedures performed in accordance with manufacturer's protocol | ||
Her2 | Polyclonal | Dako | 1:2000 | 6′ at 95°C, pH 6 | Human breast carcinoma |
TGFα, transforming growth factor α.