Surgical specimens of fresh, frozen breast tissue comprising breast tumours (n = 113) and normal background tissue (n = 30) were collected during surgery. Information was available on the Nottingham Prognostic Index (NPI), grade of tumour, degree of nodal involvement and clinical outcome for all patients with a mean follow up period of 72.2 months. The expression and levels of expression of Trio, Vav1 and TIAM-1 were analysed using RT-PCR and real-time quantitative PCR respectively.
RNA extraction and RT-PCR
RNA was isolated from breast cancer cell and tissue lines using a standard RNA-zol procedure, as we previously reported [6]. For RT-PCR, cDNA was synthesised in a 25 μl reaction mixture, as described in the manufacturer's protocol (ABgene Reverse Transcription System, ABgene, Surrey, UK). The cDNA obtained was amplified by a standard PCR mixture (as supplied in Pre-aliquoted Reddy-Load Mix, Advanced Biotechnologies). Cycling conditions for the 25 μl reaction mix were 94°C for 4 min, followed by 36 cycles of 94°C for 15 s, 55°C annealing for 15 s and 72°C for 30 s, followed by a final extension of 7 min at 72°C. The products were visualised on a 0.8% agarose gel following staining with ethidium bromide.
Quantitative-PCR analysis
The Q-PCR system used the Amplifluor™ Uniprimer™ system (Intergen Company Oxford, UK) and Thermo-Start® (ABgene, Epsom, Surrey, UK) [5, 6]. Specific primers were designed by the authors and manufactured by Invitrogen (Invitrogen Life Technologies, Paisley, Scotland, UK). Using the Icycler IQ system (Bio-Rad, Hemel Hempstead, UK), which incorporates a gradient thermocycler and a 96-channel optical unit, the plasmid standards and breast cancer cell cDNA were simultaneously assayed in duplicated 10 μl reactions as follows: Q-master mix (5 μl), forward primer (0.3 μl), probe (0.3 μl), reverse primer (0.3 μl), plasmid, (internal standard) or specimen cDNA (4 μl), water (0.1 μl). Q-PCR conditions were as follows: enzyme activation at 95°C for 12 min, 1 cycle, followed by 60 cycles of denaturing: 95°C for 15 s; annealing: 55°C for 40 s; extension: 72°C for 25 s. Using purified plasmids as internal standards, the level of each molecule cDNA (copies/50 ng RNA) in the breast cancer samples was calculated. Q-PCR for β-actin was also performed on the same samples, to correct for any residual differences in the initial level of RNA in the specimens (in addition to spectrophotometry). The products of Q-PCR were verified on agarose gels. Primer pairs for Q-PCR were as follows: Trio F1 (5'-accgttgttcttagatgtcg); Trio ZR (5'-actgaacctgaccgtacaggagatgctgtagtgaccat; Vav1 F1 (5'-agtctctggacaccacctt); Vav1 ZR (5'-actgaacctgaccgtacaccaaaatactttgtgcttcc); TIAM-1 F1 (5'-ctttaagaagaaacgctccca); TIAM-1 ZR (5'-actgaacctgaccgtacacttggctcagatcagagagt).
Immunohistochemistry
Frozen sections of breast tumour and background tissue were cut with a cryostat at a thickness of 6 μm. The sections were mounted on super frost plus microscope slides, air dried and then fixed in a mixture of 50% acetone and 50% methanol. The sections were then placed in "Optimax" wash buffer for 5 – 10 minutes to rehydrate and incubated for 20 mins in a 0.6% BSA blocking solution and probed with either Trio (sc-6060) or Tiam-1 (sc-872) primary antibody (Santa Cruz Biotechnologies Inc., CA, USA). Following extensive washings in buffer, sections were incubated for 30 min in the secondary biotinylated antibody (Multilink Swine anti-goat/mouse/rabbit immunoglobulin, Dako Inc.). Following washings, Avidin/Biotin Complex (Vector Laboratories) was applied to the sections followed by extensive washings. Diamino benzidine chromogen (Vector Labs) was then added to the sections which were incubated in the dark for 5 min. Sections were counterstained in Gill's Haematoxylin and dehydrated in ascending grades of methanol before clearing in xylene and mounting under a cover slip.
Statistical analysis
Statistical analysis was performed with MINITAB version 11.2 (Minitab Inc., State College, PA) using two sample Student's t-tests.
