RNA Extraction and Protein Expression Analysis of Bladder Tumour Samples

Eighty-eight tumour blocks from pre-radiotherapy transurethral resection of bladder tumour (TURBT) samples collected from 2002 to 2009 were identified as being suitable for this project. Patients were treated with radical radiotherapy for transitional cell carcinoma of the bladder at the Leeds Cancer Centre, West Yorkshire, UK, as per Choudhury et al [2 (link)]. An area of each formalin-fixed paraffin-embedded (FFPE) block was identified based on the matched haematoxylin-and-eosin (H+E) slide, where more than 70% of cells were tumour cells and the area appeared homogenous, to ensure that any core taken was representative of what was seen on the H+E slide. Four cores of tissue were taken from the identified area using a 0.6 mm tissue microarray corer (Beecher Instruments Inc). Total RNA was isolated using the HP RNA paraffin kit (Roche Diagnostics Ltd) according to manufacturer's instructions, except that cores were lysed for 3 days, quantified by Nanodrop and 200 ng used in a 20 μl Taqman reverse transcription reaction (Applied Biosystems). Whole mount sections were stained for MRE11 and the area scored for percentage positive cells and intensity score as per Choudhury et al [2 (link)].