Experimental Methods Anna Fahlgren, Phd Associate professor in Experimental Orthopaedics
What is experimental Methods?
Experimental Methdology
Experimental Methdology
The Formal Hypothesis The precise expression of a predicted relation between or among events; a testable idea.
Research question and Hypothesis
Research question and Hypothesis Q: Why does the zebra has different stripes pattern in different living locations?
Research question and Hypothesis Q: Why does the zebra has different stripes pattern in different living locations? H: Zebras stripe pattern depends on the vegetation in their living locations.
Building your research question and hypothesis Knowledge gap: a connection to improving human health and a broad indication of what the entire proposal is going to be about. Key points of what is known and end with a gap in knowledge that your research is going to fill.
Building your research question and hypothesis Knowledge gap: a connection to improving human health and a broad indication of what the entire proposal is going to be about. Key points of what is known and end with a gap in knowledge that your research is going to fill. Long-term goal. This needs to be bigger than the specific research. Propose the overall objective and the central hypothesis for the specific project. Provide a statement of rationale that leaves the reader with a connection back to the gap.
Writing your Aims and Hypothesis Each aim will be paired with a working hypothesis Avoid descriptive aims Base your aims on your preliminary data Avoid contingent aims where one aim has to work as you anticipate before you can do the next one. Make sure that all aims relate to the overall/ central hypothesis
Writing your Aims and Hypothesis Go back and see if your aims hang together with the knowledge gap, longterm goal, overall objective and the central hypothesis
Writing your Aims and Hypothesis On the basis of your edited statement, reflect over following questions: Does the answer to this problem have the potential for providing important, relevant answers and information?
Writing your Aims and Hypothesis On the basis of your edited statement, reflect over following questions: Does the answer to this problem have the potential for providing important, relevant answers and information? Will the results be more than a simple exercise in gathering information, answering a yes/ no question, or making a simple comparison?
Writing your Aims and Hypothesis On the basis of your edited statement, reflect over following questions: Does the answer to this problem have the potential for providing important, relevant answers and information? Will the results be more than a simple exercise in gathering information, answering a yes/ no question, or making a simple comparison? Is the problem focused enough to be doable, or is it too broad in scope (e.g., attempts to research too much across too large a geographical area or too larg population)?
Writing your Aims and Hypothesis On the basis of your edited statement, reflect over following questions: Does the answer to this problem have the potential for providing important, relevant answers and information? Will the results be more than a simple exercise in gathering information, answering a yes/ no question, or making a simple comparison? Is the problem focused enough to be doable, or is it too broad in scope (e.g., attempts to research too much across too large a geographical area or too larg population)? Is this really why I want to investigate?
Making a hypothesis General hypothesis Water levels affect the amount of lice by rainbow trout. Directional hypothesis Rainbow trout suffer lice when water levels are low. Measurable hypothesis If water levels are low then Rainbow trout will suffer more lice because there is less oxygen in the water.
What is your Aim and hypothesis!
The experimental Study
Experimental Methdology
What is an experiment?
What is an experiment?
What is an experiment?
What is an experiment? The experimental study is a systematic and scientific approach to research in which the researcher manipulates one or more variables, and controls and measures any change in other variables.
Model organism A model organism is a species that has been widely studied, usually because it is easy to maintain and breed in a laboratory setting and has particular experimental advantages.
Model system Yeast Cell line Cell culture Organotypic culture Organ Animal Human Weakness? Strength?
Experimental Method plan Ideas generated from previous research! Hypothesis testing A good scientific design makes it easier to write and publish.
Subproblem What experimental groups (how many) should be included? What is the best way to choose a sample/ subject? How large should a representative sample of a population be? What instruments or method should be used to gather data? How do I find the sub problems within the main problem?
Treatment group A group in which the value of the independent variable is manipulated or changed.
Control Group A group in which that values of the independent variable is either neutral or unchanged.
Controls - Precision matching A technique in which pairs of identical subjects are randomly assigned to one or two treatment conditions. The proposed causal relationship/model being examined allows for the probabilistic assumption that the result is generalizable. Other matched controls (depending on the sampling technique used for the study).
Treatment vs control groups?
What is the best way to choose a sample/ subject? Subject sampling Random or convenience sampling Treatment and Control groups Matched controls? Sham? Correct Control! Number of subjects in each group How this number was selected
Sampling procedure Sampling groups correctly is especially important when there are more than one condition in the experiment. One sample group often serves as a control group, whilst others are tested under the experimental conditions.
Subject sampling Convenience sampling - Selection of a group from which the sample is drawn.
Subject sampling Random sampling procedure a procedure in a sample is drawn from a population of values based on some random principle; this prevents systematic biases in the sample.
What groups are present in your study? How did you select these?
Confounding variables Uncontrolled sources of influence which can lead to misinterpretation of experimental findings. Try to list confounding variables that can appear in your experiments and make exclusions criteria.
Single and double blind experiments
Sample size
Pilot test? How large should a representative sample of a population be?
Type I and type II error A Type I error is often referred to as a 'false positive', and is the process of incorrectly rejecting the null hypothesis in favor of the alternative. A Type II error is the opposite of a Type I error and is the false acceptance of the null hypothesis. A Type II error, also known as a false negative, would imply that the patient is free of HIV when they are not, a dangerous diagnosis.
Power Power refers to the probability of avoiding a Type II error, or, ability of your statistical test to detect true differences when they are there. To low power may suggest an effect that is not there To high power is a waste of resources
Power and sample size calculator your sample size the effect size you want to be able to detect (usually medium) the Type I error rate (alpha, usually.05) the variability of the sample. Power is usually specified at 0.80, that is, 80% likely to be right. http://biomath.info/power/prt.htm
Power and sample size calculator
In conclusion: The number of subjects (sample size) depends on: The variation in the variable studies The minimum difference that is to be shown The statistical method to be used for analysis of the results The type I error agreed in advance The type II error agreed in advanced
Statistical methods Pilot test Power of the study Study design Primary (main) variable
The experimental Design Within subject design a design in which participants are assigned to more than one experimental condition. Between subject design a design in which participants are assigned to only one experimental condition.
Repeated Measure Design
Repeated Measure Design
Factorial Design Control Surgery Drug X At least two levels for each factor Control
Variables Treatment conditions or factors (independent variables) Independent variables are conditions under control of the researcher and typically manipulated in an experiment. As independent variables they lead to changes in the dependent variable. Identify the dependent variable The variable that is influenced by the independent variable.
Choosing the primary variable Alternative 1 Alternative 2 Bone mass Bone mass Number of osteoclasts Gene expression Protein expression Serum markers Number of osteoclasts Gene expression Protein expression Serum markers
Comparing many variables If you test 20 hypothesis, one of them should show p 0.05 just by chance. If you test 20 000 genes on a microarray. Correct p-values for multiple comparisons Specific algorithm or define your own threshold False discovery rate, Fold change Technical and biological replicates Correlation or cause?
Material and Methods What instruments or method should be used to gather data? Describe the instruments Validity and reliability Describe the material used for the study Describe material Discuss if a pilot test was used
Experimental Methdology
Visualize data
Visualize data
Data and their characteristics Explore different way to look at the data Normal distribution Descriptive statistics Variation Correlation Study different categories of data by figures
How did you select the group size? How are you visualizing your data?
Trouble shouting negative results Compare the right groups directly (remember your research question and hypothesis). Primary (main) variables! Positive and negative controls? Difference between two groups: Absence of evidence is not evidence of absence!
Limitations
Limitation of the study State limitation to strengthen your results
Experimental Methdology
Interpretation of data
Hypothesis and interpretation of results Identification of results Control measurements Confounding variables Confirm of abandon one or more assumptions and hypothesis Create new hypothesis!
Animal studies in biomedicine
Animal studies Not possible to do everything in humans Ethics Tool-box (genetic modifications) High level of control of your study participants -e.g age, sex, environment, food Are models valid?
Choice of species How related/ similar to humans? How easy to work with? E.g requirements, behavior High throughput? Time? Economy? Species specific advantages with systems
Inbread strains Reduces variability (genetic almost identical) Choice of strain matters Translate to humans? Can use various form of paired design Pairing: you loose statistical power Advantageous if individual variability is huge
Genetic interventions How do you know that you hit your target well enough? Validation (RNA, protein, activity) How do you interpret negative results? Controls? Biologically compensations
Pharmacologic interventions Driven by your hypothesis! Dose response How specific is the drug Replicate using another drug Replicate by KO? Drug inefficient in KO?
Cell-culture experiments Cell line Primary cells Morphology Experimental conditions Intepretation of results
Discussion examples
Example I Choice of methodology Should be able to answer your research question and hypothesis Chemicals Starvations Flox mice
Example II Surgery Sham Treatment Placebo
Intepretation of data
Critical Science
Fake Experimental Research?
Take home message!
Take home message! Experimental Methodology
Take home message! A good research planning make it faster to publish your results!