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
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
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