28 Jun MOLECULAR TECHNIQUES IN CANCER RESEARCH
molecular techniques in cancer research
Regulatory micro RNA molecules (miRNA) were discovered in C. elegans but it has become apparent that these molecules regulate gene expression in higher organism by a relatively conserved mechanism. Usually, they down regulate gene expression. Significantly, it has become known that miRNAs can act as oncogenes and tumour supressors.Six confirmed late stage cancerous tumour biopsies of the same tissue origin are available to you as well as samples of adjoining normal tissue from each. Also, you have a diverse set of normal and cancer cell lines of the same tissue origin available in your “toolbox”.
(1) Describe how you could go about identifying aberrantly expressed miRNAs in the tumours. Give due consideration to important aspects of the experimental design, methods and overall work-flow. Highlight any potential shortcomingof your approach(s) and how you would verify/confirmthe initial findingsof your search strategy (25 marks).
(2) You confirm the existence of two novel miRNAs, one that is up-regulated and one that is down-regulated in the cancer you are studying. Describe strategies for direct identification of proteins that are regulated by these miRNAs in a biological system. Be mindful of important aspects of the work-flow and be critical when considering advantages and disadvantages of the strategies you describe. (25 marks). How could you further substantiate and verify your findings (10 marks)?
(3) During the analysis of one of the miRNAs, you observe that overexpression caused the target cells to shrink, round up and detach from the plate. You suspect the induction of apoptosis. Describe tests that would confirm the induction of apoptosis (15 marks).
(4) Of the genes whose expression is modulated by the miRNA that induces apoptosis, your analysis from mining the biological databases and tools for in silico protein analysis identifies a candidate nuclear protein “APR” likely to be involved in the apoptotic response. Describe two distinct experimental strategies to identify directly proteins that may interface functionally with “APR” (15/25 marks). As in (1), give a critical appraisal of the relative advantages and disadvantages of each strategy (10/25 marks).
Maximum 2500 words for the complete answer (questions 1-4). In all cases you should justify clearly your response and cite relevant material where appropriate. Citations should not be a substitute for including key information in your answers; they should indicate the extent of your reading and ability to identify key papers. The use of diagrams is encouraged (maximum one original, clearly legible, half-page per question).
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