试验设计基本方式-国立成功大学基础医学研究所.PDF

實驗設計基本方式 賴明德 成功大學醫學院基礎醫學研究所 20010/11/29 How to design experimental approaches from an idea? • Define the question: an experiment should address a hypothesis by answering one or two specific questions. Be sure not to pick questions that it is not possible to answer. Set up an experiment • 將問題以明確陳述句表示。 • 試用圖解表示問題。 • No experiments should be undertaken without a clear preconception of the forms its result might take. (you will include adequate control and have well-designed experiment by this way) • Design the experiment: experimental variables and controls. Controls • There are two types of controls: experimental control and treatment control. • Treatment controls are positive and negative, and show you whether the experimental handling of the cells has elicited an effect. • Experimental controls tell you if the basic experimental procedure are working properly. For example, transfection efficiency measured by LacZ cotransfection, or E. coli transformation efficiency. Design an experiment: How many controls do I need ? • Internal control is absolutely required. Actin in western control, GAPDH in RT-PCR. • Negative control is required. • To investigate whether AKT is required for c-fos induction. Cells cotransfected with AKT siRNA and c-fos luciferase reporter, the luciferase is measured. pCMV-galactosidase is used as internal control. The scrambled Rnai is used as negative control. • Positive control is highly recommended when the result is positive. Positive control is required when the result is negative. • Cells treated with tunicamycin (ER stress inducer), and KCC4 ion transporter is measured. The GRP78 as a positive control for the induction of ER stress. Exampl