Illumina HiSeq 2500 paired end sequencing; RNAseq of prostate biopsy tissue from prostate cancer patients on active surveillance over 12 months on different diets (ESCAPE trial)
RNAseq of prostate biopsy tissue from prostate cancer patients on active surveillance over 12 months on different diets (ESCAPE trial)
Forty-nine men with a clinical diagnosis of low- or intermediate-risk organ-confined prostate cancer underwent two transperineal template biopsy procedures at 12-month intervals. All patients had an American Society of Anaesthesiologists (ASA) co-morbidity score of 1 or 2, which reflects the health status of men eligible for active surveillance. Of the 25-35 cores obtained each at T0 and T12, three were reserved for next generation sequencing (NGS), three were used for targeted and non-targeted metabolite analyses, two were snap frozen and the remainder underwent routine histopathological examination.   ESCAPE study design. The ESCAPE study involved the recruitment of 49 men with of low- or intermediate-risk organ-confined prostate cancer underwent two transperineal template biopsy procedures at 12-month intervals. The protocol was approved by the Quadram Institute Bioscience Human Research Governance Committee and subsequently by the National Research Ethics Service (NRES) East of England - Cambridge South Research Ethics Committee (REC ref: 13/EE/0110). ESCAPE has been adopted on the National Institute for Health Research Clinical Research Network (NIHR) Clinical Research Network (CRN) Portfolio in May 2013 (Study ID: 14482). The trial has been registered on ClinicalTrials.gov (NCT01950143), (ii) International Clinical Trials Registry Platform (ISRCTN40496794), and (iii) Clinical Trials database Cancer Research UK (http://www.cancerresearchuk.org/cancer-help/trials/a-trial-looking-effect-sulforaphaneprostate-cancer-escape.  Immediately prior to the study, the volunteers completed a comprehensive seven-day diet diary to assess their habitual diet. During the 12-month interval, the volunteers consumed their habitual diet supplemented with one 300 ml portion of a broccoli-based soup per week. The soups were otherwise identical but were manufactured from one of three broccoli Myb28 genotypes, delivering a mean ± standard deviation for standard broccoli soup of 8.4 ± 2.8, beneforte soup of 280 ± 8.8 and beneforte extra soup of 452 ± 10.6 µmoles glucoraphanin per 300 ml portion. Further diet diaries were completed at six and twelve months.  Diet data from both habitual diet and the additional soups were integrated and analysed through DietPlan6 combined with additional data on chemical composition of cruciferous and alliaceous vegetables obtained from analyses of vegetables purchased in the retail outlets in the localities of the volunteers. Disease staging refers to WHO grade for prostate carcinoma.  Two cores per patient weighing between 3 and 10 mg in total were homogenised with a QIAGEN TissueRuptor before total RNA was extracted with the QIAGEN RNeasy Mini kit. The resulting RNA was quality checked with an Agilent Bioanalyzer and samples with RIN values higher than 7 were further processed. Samples were subjected to ribodepletion with the Ribo-Zero Magnetic Gold rRNA Removal Kit (Illumina). Libraries were constructed with the Illumina barcoded TruSeq kit.