Quantitative Real-time Reverse Transcription-PCR (RT-PCR)
Breast cancer samples were obtained in the Edinburgh Breast Unit (Western General Hospital, Edinburgh) with patients' informed consent and ethical committee approval. Biopsies were snap frozen and stored in liquid nitrogen until RNA extraction. Before RNA extraction the frozen tissue was defrosted and stabilized in RNA-later-ICE reagent (Ambion). Total RNA was extracted with RNeasy-mini columns (Qiagen).
Amount and purity of RNA were evaluated by spectrophotometry. RNA integrity was confirmed by agarose gel electrophoresis. Total RNA was isolated from tumour and normal tissue using Qiagen RNeasy kits (Qiagen). RNA was treated with DNase using DNAfree kit (Ambion, Cambridgeshire) and 1 μg was used for RT-PCR using Retroscript kit (Ambion, Cambridgeshire, UK). TaqMan Assay-on-Demand gene expression pre-designed primer and probe sets from Applied Biosystems, Cheshire, UK were used for EEF1A2 (Assay # Hs 00157325 ml) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control; Hs 99999905 ml). In a 10 μl reaction volume per well of a 394-well plate, 0.5 μl of primers, 5 μl of TaqMan PCR Master Mix, no AmpErase UNG 10×, and 4.5 μl of diluted cDNA were added (Applied Biosystems, Cheshire, UK). Real-time RT-PCR and the quantification of RT-PCR products were performed and the products analyzed using an ABI Prism 7900HT Sequence Detection System, and the appropriate software (SDS3.1) according to the manufacturer's instructions (Applied Biosystems, Cheshire, UK).
Protein lysates from cell lines were prepared using previously published protocols . Western blot analyses were carried out using standard protocols. The blots were incubated with primary anti-eEF1A2 rabbit antibody and primary anti-eEF1A1 sheep antibody diluted 1:200 in blocking solution, as well as primary anti-glyceraldehyde-3-phosphate dehydrogenase polyclonal mouse antibody (Chemicon International, Hampshire, UK) diluted 1:10000. Blots were then incubated in the appropriate horse radish peroxidase conjugated secondary antibody (Dako Cytomation, Cambridgeshire, UK) at 1:500. Detection was performed using enhanced chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK).
Specimens of normal and cancerous tumours were obtained with informed consent and local ethical committee approval from patients undergoing surgical treatment at the Royal Infirmary of Edinburgh and Western General Hospital, Edinburgh. A breast tumour histoarray (CB2) produced by SuperBioChips (AMS Biotechnology, Oxfordshire, UK) was also used. Formalin fixed, paraffin embedded sections of human normal tissue and tumour tissue were deparaffinized with xylene, rehydrated, treated with picric acid and microwaved in citric acid pH6. Slides were blocked in a 1:5 dilution of sheep serum for 30 minutes at room temperature. Primary anti-eEF1A2 rabbit antibodies were used at a concentration of 1:10 diluted in PBS, for 40 minutes at room temperature and secondary goat anti-rabbit IgG biotin conjugated antibody (Dako Cytomation, Cambridgeshire, UK) was used at 1:200 at room temperature for 30 minutes. Slides were incubated with StreptABC complex/HRP (Dako Cytomation, Cambridgeshire, UK) at room temperature for 30 minutes and in diaminobenzidene (Sigma Fast DAB, Sigma, Dorset, UK) for 2 minutes at room temperature. Finally slides were counterstained in haematoxylin, dehydrated and mounted in pertex.
Immunohistological scoring methods
The breast tumour sections and normal breast sections (CB2, SuperBioChips, AMS Biotechnology, Oxfordshire, UK) were scored as weak, moderate and strong staining for eEF1A2. Weak staining was considered as background since this level of staining is seen in normal tissue. Stromal tissue was negative in all cases. Blind scoring was carried out by two independent researchers. Two slides were analysed, representing different levels within tumours, and each of these was stained with a different antibody to eEF1A2. Almost perfect correlation was seen between the two slides.
Fisher's exact test was used to test for associations between negative and weak eEF1A2 expressing tumours or moderately and strongly overexpressing tumours with ER positivity. For breast tumour Quantitative Real-time RT-PCR data, a two-sample t-test allowing for difference in variance between the two samples was used to test the difference between the mean standardised quantity of RNA for the ER-positive and ER-negative groups. P values that were less than or equal to 0.05 were considered significant.