blanching prior to freezing denatured plant myrosinase, thus the levels of SF and IB derived from the high glucosinolate broccoli diet would be similar to or lower than those obtained from fresh broccoli with functional myrosinase. Levels of indole glucosino- lates were similar in both high glucosinolate and standard broccoli. Compliance monitoring and dietary assessment Volunteers completed weekly tick sheets during the 12-month intervention period to identify when the portions of vegetables were eaten. Every two weeks, volunteers were contacted by telephone and asked about adherence to the diet. A seven-day estimated food intake diet diary was completed by volunteers at baseline and after six months using household measures as an indication of portion size. Food intake from the diaries was inputted into Diet Cruncher v1.6.1 (www.waydownsouthsoftware. com/) and analyzed for differences in nutrient composition between the two intervention groups at baseline and six months after intervention. Genotyping Genomic DNA was extracted from whole blood or from tissue samples using Qiagen QIAamp DNA minikit with RNase treatment according to the manufacturer’s instructions (http:// www.qiagen.co.uk/). GSTM1 (NM_000561) genotype was deter- mined using a real-time PCR procedure based on Covault and colleagues, using gene specific primers and probe and quantified relative to a two-copy gene control, a region in IVS10 of the breast cancer 1, early onset (BRCA1, NM_007294) gene [21]. Primers and probes were designed using Applied Biosystems Primer Express (http://www.appliedbiosystems.com/) and are given with PCR conditions in Table S1. Data were analyzed with Applied Biosystems Absolute Quantification software. RNA extraction and microarray hybridisation Total RNA was isolated from the TURP tissue bank samples and the TRUS-guided needle biopsies from the volunteers with the use of QIAGENH RNeasy mini kits according to the manufacturer’s instructions (http://www.qiagen.co.uk/). The quantity of resulting RNA was measured using a spectrophotom- eter (Beckman). The RNA quality was determined using the Agilent 2100 Bioanalyzer (http://www.agilent.co.uk/). RNA samples from TURP biopsies of benign and malignant prostates and from TRUS-guided biopsies from both subject groups (peas and broccoli) at baseline, and at six and 12 months after intervention were hybridized onto Affymetrix Human U133 Plus 2.0 microarrays (http://www.affymetrix.com/) by the Nottingham Arabidopsis Stock Centre (NASC, http://arabidopsis.info/). Double-stranded cDNA synthesis and generation of biotin-labeled cRNA were performed according to the manufacturer’s protocol (Affymetrix, http://www.affymetrix.com/). The final cRNA was checked for quality before fragmentation and hybridization onto the arrays. One of the 22 volunteers was diagnosed with prostatic adenocarcinoma at the study baseline biopsy and was removed from the study. Eleven samples from the baseline biopsies, two samples from the six-month biopsies and three samples from the 12-month biopsies did not produce good quality RNA and/or sufficient cRNA and were not hybridized. In addition, one volunteer showed prostatic adenocarcinoma at the six-month biopsy; subsequent samples were removed from the study. Fluorescence intensity for each array was captured with a GeneChipH Scanner 3000 7G. Affymetrix GeneChipH Operating Software (GCOS) was used to quantitate each U133 Plus 2.0 array. Microarray data in this paper are compliant to the Broccoli & Signalling Pathways minimum information about a microarray experiment (MIAME) criteria and are deposited at Array Express (http://www.ebi.ac. uk/microarray-as/aer; Accession Number E-MEXP-1243). Microarray data analysis Raw data files (CEL) were loaded into the DNA-Chip Analyzer software (dChip, http://biosun1.harvard.edu/complab/dchip/, build date September 2006) for normalization, generation of expression values and statistical analysis. Following normalization using the Invariant Set Normalization method, probe expression levels were calculated using the PM-only model. To identify genes that were changing between groups, different two-tailed P-value thresholds were applied calculated by Welch modified two-sample t-test in dChip. Paired or unpaired t-tests were performed as appropriate. To correct for multiple testing, False Discovery Rate (FDR) was estimated by permutation in dChip and the median of 100 permutations reported for each of the comparisons (1000 permutations on selected samples had little effect on FDR calculations). Unsupervised clustering was performed on benign and malignant samples using 1-Rank correlation as distance metric on a gene list of 3697 probes. These probes satisfied two criteria: first, that the coefficient of variation (CV) was between 0.5 and 1000; and secondly, that the percentage of Presence calls was more than 20% across all TURP benign and malignant samples. For the purpose of sample classification, 19 laser-capture microdissected (LCD) epithelial cell microarrays (GEO Accession: GDS1439, http://www.ncbi.nlm.nih.gov/geo/) and 32 TURP benign and malignant microarrays were normalized together and model-based expression was calculated as described above in dChip. The LCD samples were derived from six benign prostate tissue samples, five clinically localized primary prostatic adeno- carcinoma samples, two replicates of the five primary cancer samples after pooling, four metastatic prostatic adenocarcinoma samples and two replicates of the four metastatic prostate cancer samples after pooling [22]. Classification of the LCD epithelial cell samples was then performed using linear discriminant analysis (LDA) based on the TURP benign and malignant samples as training samples. LDA was performed using 442 probes that had higher than 100 units difference in signal intensity between TURP benign and malignant samples and were significantly different at P#0.01 by Welch modified two-sample t-test.To identify pathways that are the most over-presented in the lists of differentially expressed genes, functional analyses using MAPPFinder and GenMAPP v2.1 were performed (http://www.genmapp.org/). Incubation of peptides with isothiocyanates Incubations of SF or IB with bovine insulin (P01308, Sigma- Aldrich), recombinant human epidermal growth factor (EGF, P01133, R&D Systems, http://www.rndsystems.com/) and re- combinant human transforming growth factor beta 1 (TGFb1, P01137, R&D Systems) were performed in sodium phosphate- buffered saline solution (pH 7.4) or human blood plasma at 37uC for 0.5–24 h. Plasma was pre-treated by ultrafiltration to remove high molecular weight proteins (Microcon Ultracel YM-30 filter, MWCO 30,000). Samples were either analyzed directly by LC- MS/MS or by LC-MS/MS analysis of tryptic digests of gel electrophoresis bands. Direct LC-MS/MS analysis of peptides incubated with isothiocyanates The LC system used was a Shimadzu series 10AD VP (Shimadzu, http://www.shimadzu.com/). The column was an ACE 300 C18, 15062.1 mm (5 mm particle size) used at 40uC. PLoS ONE | www.plosone.org 4 July 2008 | Volume 3 | Issue 7 | e2568