the cell pellet washed twice in PBS before freezing at -80oC until use. MicroRNA extraction from the washed cell pellet was performed as highlighted in chapter 2.2.5. Initially, comparison of RNA yield was achieved using matched samples collected into an empty container or one containing 100% volume of the RNase inhibitor, namely diethylpyrocarbonate (DEPC). Fifty consecutive urine samples were collected to determine the value of the stabilising agent DEPC. The dilution with this carcinogenic and toxic agent was done in a ratio of 1:1 using standard universal precautions including the utilisation of a fume cupboard. To determine microRNA stability in we split 25 samples into three fractions (50mls for each) and separately extracted RNA in the form of two reference genes (RNU48 and RNU44) and three microRNAs (15b/135b/1224-3p) from an immediately processed sample, following 3 cycles of freeze-thawing (-80oC to room temperature) and following storage at room temperature for 48 hours. Chapter 3 – MICRORNA EXPRESSION IN UROTHELIAL TISSUES Evidence to identify any roles for changes in microRNA expression in bladder cancer was very limited at the time of commencement of this study. Furthermore there was no evidence suggesting that microRNA could be an additional epigenetic tool supporting the theory that the high-grade and low-grade bladder
had pTis disease, 13.5% (n=7) had pT1 disease and 34.6% (n=18) patients had pT2-4 disease. With regards to the 20 normal urothelial samples, the male to female ratio was 9:1 (18 versus 2) with the same age range as the bladder cancer samples of The 10 samples that were from patients who had no history of bladder cancer were obtained from patients who underwent trans-urethral resection of prostate (n=4) and radical prostatectomy (n=6). The 10 macroscopically normal looking samples that were collected from patients with bladder cancer were obtained from patients that either underwent a radical cystectomy (n=7) or a trans-urethral resection of bladder tumour (n=3). Overall follow-up of bladder cancer patients incorporating all grades of disease ranged from 2-93 months. This represented a mean follow up of 43.4, 57 and 23.5 months for NMI, high-grade NMI and invasive disease respectively. Bladder cancer Normal urothelium Low-grade NMI High-grade NMI Invasive Cancer history Total 22 12 18 10 10 Gender Male Female 14 8 12 0 14 4 10 0 8 2 Age Mean Range 75 56-83 71.9 55-81 71.4 46-90 67 54-81 66.4 46-90 Source TURP RP TURBT RC 4 6 3 7 Grade 90.
brought into clinical practice in a three worded statement – “Validation, validation, validation.” Due to only using 63 bladder cancer urine samples we did not perform an analysis of associating various grades and stages of bladder cancers to specific urinary microRNA levels. This study did have a balanced split between low-and high-grade disease of 53% and 47% respectively, which was not the case for other similar studies. Further studies with an increased number of urines samples may allow the opportunity to allow disease stratification with the various grades and stages bladder cancer. An argument for not performing differentiation between the identification of low-and high-grade disease is that we targeted our study to be a diagnosis of bladder cancer universally and not just a specific subset of disease. The diagnosis of low-grade bladder cancer is pathologically and clinically of a lower priority than it’s high-grade counterpart however it must always be appreciated that a significant proportion of low-grade disease will to develop into the high-grade form. An obvious criticism and weakness with regards to the diagnostic data is the specificity value of 51%. This was at the opportunity cost of a high sensitivity value 94.1%. Of note when implementing a urinary test to stratify the need for a cystoscopy, it is sensitivity (and hence the reduction of false negative tests) that takes priority over specificity. This study did not incorporate the analysis of post-operative urine samples for those undergoing the resection of their tumours. However it can be argued that