Download Experimental Methods

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
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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
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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
Surgery
Control
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 2
Alternative 1
Bone mass
Bone mass
Number of
osteoclasts
Gene
expression
Protein
expression
Serum
markers
Gene
Number of
osteoclasts 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
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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!
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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
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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!