Unit 1 Flashcards
What does gene expression measure?
- Not DNA (consistent)
- Either protein or RNA levels
What method needs small amounts of mRNA to measure epxression?
qPCR
Molecular clock
genetic network of different clock gene proteins that general primary and secondary feedback loops inside the cell nucleus
Steps of the Period Molecular Clock
- CLOCK/BMAL1, when active, increases PER and CRY expression because it binds to its promoter
- PER and CRY proteins feedback to inhibit and stop CLOCK/BMAL1-driven expression of PER and CRY
- As levels of PER and CRY go down, CLOCK/BMAL1 bind to promoter and increase PER and CRY transcription again
How long does the period molecular clock take?
24 hours
Are we looking at circadian rhythm in the experiment?
No!
- If we measured at two different time points, then yes
- Because we are just looking at PER expression, it isn’t necessarily the circadian rhythm
Suprachiasmatic Nucleus (SCN)
- In hypothalamus
- Master pacemaker
- Retinohypothalamic tract simulates wave of cellular activity in SCN due to light
- This pattern is expressed to other cells in the body with clock genes
What do clock genes regulate?
Circadian rhythms (which can be a lot more than just sleep!)
Common causes of circadian rhythm disruption
- Behavioral (jet-lag, shift work)
- Social (blue-light (slow wavelength light effect)
- Medical (insomnia, blindness)
True or False: if the experimental design is flawed, the data are not valid.
True!
How to recognize pseudoscience?
- Lack of credible sources
- Illogical leaps
- Reference to old wisdom
- Accrediting the author with many things
- Attempting to sell you something
What is the purpose of a pilot study?
To due a small scale, preliminary study before a larger scale study to determine overall feasibility of the larger study, design parameters, possible adverse events, and others to not waste time and money
Best way to pick a question
Start broadly, then refine and focus to a testable hypothesis
Falsifiability
Show that your hypothesis can be false
Operationalized hypothesis
Hypothesis written in terms of the operations and procedures used to test it
Internal validity
How reliable and replicable your results are and if you can determine a causal relationship between variables
Reliability
Measurements give similar results each time they are repeated under the same conditions
Replicable
Results are seen when the experiment is repeated and similar results are obtained
External Validity
How well the research can be applied/generalized to other populations and settings
Ecological Validity
How well the research mirrors conditions in the real world
Predictive Validity
How well your measures can predict important outcomes
True or False: you can use pipettes outside the range that is indicated on the side
False
Pipette ranges for orange pipette tips
0.1-2 microliters
1-10 microliters
Pipette ranges for yellow pipette tips
5-50 microliters
20-200 microliters
Pipette ranges for blue pipette tips
100-1000 microliters
Steps to fill pipette and dispense the liquid
- Keep pipette upright to both fill and dispense
- Press plunger down to the first stop before placing in liquid
- Place tip in a small distance below surface of liquid and slowly release plunger. Leave in long enough for liquid to enter tip, then withdraw tip
- Dispense solution onto tube surface or into liquid. Do not dispense into air. Dispense by pressing plunger down to the second stop. With without release the plunger.
General idea behind a spectrophotometer
- White light splits into different wavelengths
- Single wavelength selected and shone through solution
- Light will be absorbed by the solution. More light is absorbed as the concentration of protein increases
- Any light not absorbed is detected and measured
At what wavelength do proteins absorb the most light?
280nm
Beer Lambert Law
Relates the absorption of light to the properties of the material through through which the light is traveling, so assuming you have a pure solution of protein, you can use the law to determine the concentration
Serial dilutions
Mix half and half, then add some of the mix to the next tube
Relative concentrations
The amount of solution added with a relative concentration
How many microliters are added to spectrophotometer with the serial dilutions?
3
How to graph the spectrophotometer and relative concentrations
Relative on X, actual on Y; should be linear
Concentration of the solution _____ each time a dilution is made
Halves
Observational studies
- Non-experiment
- Neuroanatomy studies
Case studies
-Non-experiment
- Thorough analysis relating to a single object or participant, such as a person with a unique type of brain damage
What does an experiment involve?
An independent variable (manipulated by research) and a dependent variable (measured variable)
To see if the IV is affecting the DV, you must appropriately ______ the IV
control
Independent Samples design
2 separate groups of subjects and each group has a variation of the IV
Levels of IV
Different variations of the IV, for example multiple doses of the same drug, plus an appropriate control treated group
Confounding variables
Change with the independent variable and outcome, and should be avoided!
Controlled variables
Variables other than the independent variable: the only variable that should be different between the two groups is the IV
Not all uncontrolled variables are _____
confounding variables
Positive Control
Checks that the procedure is working
Negative Control
Checks that the procedure isn’t giving you false positive results
Purpose of negative and positive controls
To show that the procedure is working reliably
Vehicle control
- Drug administration studies
- Vehicle = solvent for a drug (not always saline!)
- Controls for all other non-drug variables associated with drug administration
Sham Surgery Controls
- In studies where the IV involves a surgery, a sham surgery is the appropriate control
- All variables of the surgery are the same, expect the step that represents the IV
- Note: some experiments involve surgery that’s the SAME in all groups, in this case, there are no sham surgery controls
Random assignment
-Addresses the problem that there may be variables that have not been thought of and explicitly controlled for
- Randomly assigning subjects across the experimental conditions (so that variables associated with subjects are equally distributed)
Randomization of treatment
Running experimental and control groups synchronously, rather than all control subjects and then all experimental subjects
How to best randomize
Random number generator or pulling numbers from a hat!
Within subjects design
Each subject undergoes all the experimental and control conditions (often used in fMRI studies)
Matched Sample design
Subjects are matches for that variable between groups, such as a baseline difference in the dependent variable
How to conduct a matched sample design
- Pretest to measure the variable
- Rank order based on the data
- For a 2 group experiment, form pairs starting from the top two subjects
- Randomize pairs to each group
Attrition
Loss of subjects before the end of the experiment
How could attrition be bad for an experimental design?
If one group experiences more attrition than another group, it might be due to a confounding variable
Quasi Experiment
An “experiment” in which the IV is a subject characteristic (something that can’t be changed between the subjects)
- An example would be transgenic mice
Types of Transgenic Mice
Knock-Out - gene inactivated so it does not produce the protein
Knock-In - gene inserted allows for expression of a new protein or over-expression of a naturally one
Control - Wild type
Automate
Using devices or machinery for things - whenever possible, automation should be used (will control for bias)
What should you do if you can’t automate?
Have more than one person observe and rate with good reliability (consistency) between observers
Placebo
Inert substance (not the vehicle) sometimes used as a control in drug administration studies in people (not often done in animal studies)
Why do we need placebos?
- Health conditions can change over time so there can be spontaneous remission
- Response to taking a drug could not be due to its pharmacological effects alone
Placebo effect
Can be really large (referring to response to taking a drug could not be due to its pharmacological effects!)
Ethical concerns of placebo effect
-Subjects should know there is a possibility they are not getting the drug
- You do not use placebos when there is proven treatment (like NSAIDs)
Single-Blind Study
The experimenter knows which group the subject is in, but the subject does not!
Double-Blind Study
Neither the experimenter nor the subject knows which group the subject is in (best design!!)
How should you analyze data?
BLIND
Special concerns in pharmacological studies
-Important to perform a does response curve
- Log dose often shows a linear response pattern, so doses used SHOULD have a 1, 3, 10, 30, 100 pattern
Order effects
In a within subjects design, if you always run the same experimental condition first, you might have an order effect: first experimental condition influences the response to the second condition
Counterbalancing
- Helps prevent order effects
- Randomize the order of conditions
Time-Series Design
- Helps prevent order effects
- Dependent variable is measured at different times with the control or experiment condition applied between measures
- I.E. Control condition A, followed by Experimental condition B, followed by Control condition A
- Helps to control for the effect of time!
Designs in fMRI Experiments
- Blocked Design
- Event-Related Design
Blocked Design
fMRI experiments often follow this design, where experiments conditions are alternated with a rest period in between (this rest, task, rest, task)
Event-related Design
Present test stimuli for a very short period of time, with longer time in between for control condition
True or False: you can conclude brain activity caused behavioral responses in fMRI studies
False
Cause and Effect
- If all variables were controlled for and the IV was changed, we can assume the difference in variables is due to the IV or to chance (use inferential statistics!)
When is “x” necessary for “y”
If an effect y doesn’t happen when x is removed, x is necessary for y
When is “x” sufficient for “y”
If an effect y happens when x is added, with all other variables constant, x is sufficient for y
Convergent evidence
General hypothesis is supported by multiple experiments using different techniques that come to the same conclusion
Meta-analysis
Analysis of data from multiple studies on the same topic
Population
Entire collection of cases of interest
Sample
Subset of the population
True or false: if you want to compare two groups to determine if they differ, in the language of statistics, you are asking “do these two samples come from different populations”?
True
Descriptive statistics
Describe and visualize the sample data
Inferential statistics
Determine the probability that the samples are from different populations
True or false: Scientists often use the word “proof”
FALSE
Stratified Random Sampling
Ensure equal representation of groups within a population
Type I Error
-You reject the null and conclude the groups differ when they do not
- Alpha (0.05)
What is a statistically significant difference
- When a test statistic meets or exceeds the critical value determined by alpha (0.05)
Type II Error
- You accept the null when it is actually false
- Beta (also shows power (1-beta))
Power
1 - beta
What kind of sample sizes diminish the chance of making a Type II error?
bigger
Factorial designs
- Multiple IVs
- Simplest is 2x2
- Each number represents an independent variable, the numerical value represents the number of levels that variable has
How many independent variables are in a 2x2 design?
2
How many levels do each of the variables have in a 2x2 design?
2
How many groups are in a 2x2 design?
4
Tuskegee Syphilis Study
Unethical research in which African Americans with syphilis were told they don’t syphilis, didn’t get consented, were not informed that the study would not benefit them, and did not know and weren’t offered penicillin
Common Rule Policy
Regulates research studies with humans under many federal agencies
Office for Human Research Protection (OHRP)
Under the Department of Health and Human Services
IRBs are overseen by
OHRP
IRB
Group of 5 members that review all research on human participants at a certain institution
Belmont Report
3 major aspects of ethics
1. Respect for persons
2. Beneficence
3. Justice
Respect for Persons
Informed consent
Beneficence
Benefit-risk analysis, confidentiality, and protection from harm
Justice
Study the population that would benefit the most
What factors should you consider when considering animal research?
Type of experiment, type of animal, number of animals required
3Rs
Refine
Reduce
Replace
Name of the first edition of legal regulations of animal use in USA
Guide for the Care and Use of Laboratory Animals
Animal Welfare Act
Covers all warm blooded animals except for birds, rats, and mice (and farm animals used in agricultural research) - under USDA
PHS Policy on Humane Care and Use of Laboratory Animals
Research supported by the Public Health Service: covers ALL vertebrate animals
PHS Policy is overseen by whom?
Office of Laboratory Animal Welfare (OLAW)
IACUC
-IRB for animal research
- All use of vertebrate animals must be reviewed and approved before animals are used
IACUC Protocols
-Rationale for using the specific animals
- Specific procedures (including housing)
- Educational benefit
- Justification of species
- Description of pain and distress experienced by animal
Who sits on the IACUC?
- Scientists
- Non-scientists
- Vet
- Non-affiliate member
Two ways data can be unethically handled
Fraud and Bias
Fraud of data
Outright fabrication of data or unjustified conclusions, including changing or omitting data, altering equipment to achieve desired results
Bias of data
Beliefs can cause data bias if there is any subjectivity in the measure
- Mathematical errors that aren’t corrected
What is a classic example of bias in data handling?
Morton vs Gould (Morton thought race had an impact on skull size, Gould found that it didn’t)
What is the bottom line of the Gould vs Morton debate?
Our personal beliefs can bias data collection and analysis, either consciously or unconsiously
Steps to prevent bias
- Analyze all data blind to experimental conditions
- Double check all data - especially calculations
- Automate all procedures and data handling
Steps to Show Research is not fraudulent
- Keep detailed notebooks with best practices (pen!)
- Maintain files with raw data
What are two ways to quantify levels of mRNA
- In situ hybridization (anatomically specific)
- qPCR (sensitive and detects very low levels of mRNA)
Cockroach “brain”
Consists of 2 fused ganglia
Cockroach Ventral Nerve Cord
- Arises from “brain”
- Lies of ventral side
- Has more ganglia (3 in thorax and 6 in abdomen)
How many ganglia are in the thorax of a cockroach?
3
How many ganglia are in the abdomen of a cockroach?
6
True or false: the VNC is the spinal cord in cockroaches
False
General overview of VNC dissection
- Anesthetize cockroaches in ice water
- Cut off parts that can move
- Pin to dissection dish ventral side down
- Remove all of abdominal exoskeleton dorsally
- Carefully remove organs and fat using blunt dissection, working parallel to nerve cord
- Remove as much fat as possible from nerve without breaking it
- Use a bent seeker to clear the nerve cord from the ventral exoskeleton
- Hold the nerve cord with forceps and cut out a 1-2 cm piece of cleared nerve cord and place in 2 ml microcentrifuge tube
How to reduce the amount of RNA that degrades
- Work as quickly as possible when you dissect the tissue
- Always wear gloves when working with your tissue/RNA
- Keep sample on ICE
What breaks down RNA?
RNase (ribonuclease enzymes present in tissues)
Organic Extraction of RNA Overview
- Sample is homogenized in a phenol based solution, then centrifuged
- Sample separates into 3 phases
1. Lower organic phase
2. Middle phase called interphase
3. Upper aqueous phase - Organic and Interphase have everything, aqueous phase has RNA!
- Aqueous phase is recovered and RNA is collected by alcohol precipitation and rehydration
Benefits of organic RNA extraction
- Rapid denaturation and stabilization of RNA
- Good for removing gDNA
- Scalable technique
Drawbacks of organic RNA extraction
- Use/disposal of organic reagents
- Manually intensive processing
RNA Isolation by Spin Column
- Spin columns use membranes across the bottom of a small plastic column/basket
- Tissue samples are homogenized in a special buffer, added to column, and centrifuged (spin column!)
- This pulls sample through membrane and nucleic acids bind to membrane
- To get rid of DNA specific nucleic acids, the membrane is washed and degraded with DNase and then eluted appropriately
Benefits of Spin Column Method
Convenient, easy to use, good for all samples, can be automated
Drawbacks of the Spin Column Method
Spin columns can easily clog, limited binding capacity, and can retain gDNA!!!!!
What is in a lysis buffer?
Guanidine thicyanate and 1-Thioglycerol
Purpose of Guanidine thicyanate
- Lyses cells (breaks their membranes)
- Disrupts nucleoprotein complexes
- Inactivated RNases
Purpose of 1-Thioglycerol
Inactivate RNases
What do we add to the RNA tissue?
Lysis buffer
What do we do after extract RNA tissue and adding it to lysis buffer?
Homogenize and pipette 10 times (shear)
What happens after homogenizing and shearing of homogenate/lysate?
Centrifuge - pipette the solution in the middle as to not pick up fat or insoluble crud
What is added to the homogenized and centrifuged sample?
Isopropanol
General steps of the spin column (from the actual lab)
- Lyse tissue
- Bind to column with isopropanol
- Wash/Centrifuge
- DNase treat (15 minutes)
- Elute and wash RNA from column using RNase-free water
Why do we need to make cDNA?
RNA can’t be amplified, so we need to make a complementary DNA copy of the RNA to quantify in a qPCR
What is the ratio of mRNA to DNA?
1:1
Best absorbance of RNA
260nm
Why do we measure RNA concetration?
To make sure we start with the same amount of total nucleic acid so we can fairly compare the samples
A260:A280 ratio should be
2
Why should the A260:A280 ratio be 2?
Because the absorbance at 260 should be twice that at 280
What type of primer did we use?
Oligo (dT) primer
What does “oligo” mean?
Short for “oligonucleotide” - short length of DNA
What does “dT” mean?
Sequence containing only thymine molecules that will bind to a sequence of adenines in the RNA sample (the poly A tail) - makes it selective for mRNA
Other types of primers we could use
- Gene specific primer
- Random primers
Why would we use gene specific primers?
High specificity, but must rerun cDNA synthesis for every gene of interest
Why would we use random primers?
Consists of multiple short primers sot hat all RNA molecules are selected: also good for RNA without a poly-A tail
Reagents needed for cDNA synthesis
Buffer, primer, nucleotides, water, enzyme (reverse transcriptase)
Purpose of qPCR
To amplify and detect in real time a specific sequence of DNA; AKA real-time PCR
Difference between PCR and qPCR
PCR - accumulation of DNA is measured only at the end of the procedure
qPCR - accumulation of DNA is measured during and throughout the procedure
The amount of DNA _____ with each PCR cycle
Doubles
RT-PCR
Reverse transcription PCR: When RNA is not transcribed into DNA before, and is instead the first step in the actual qPCR procedure
True or false: you cannot amplify RNA
True
Two types of methods for detecting products of qPCR
- Sequence-specific probe
- SYBR Green Method
Sequence-specific Probe Overview
- A labeled fluorophore probe with a quencher, which prevents the detection of fluorophore when close by
- Taq DNA polymerase has 5’ exonuclease activity, which will degrade the TaqMan probe (quencher) in its path, releasing the labeled fluorophore!
SYBR Green Method Overview
- A fluorescent dye that interacts and fluoresces in double stranded DNA
- Binds to the minor groove of dsDNA and emits green light
What is in the qPCR reaction tube?
- Template DNA
- Primers (forward and reverse)
- Water (as needed)
(following are supplied at 2x concentration) - Nucleotides
- Buffer
- MgCl2 (cofactor of enzyme)
- Taq Polymerase
- SYBR Green
What is the VERY first step in qPCR?
Hot Start! - Denatures first strands of cDNA AND activated Taq, which is blocked by antibodies until it is heated
Three main steps of qPCR
Denature, Anneal, Extension
Denaturation
- Heat DNA so it gives two single strands of DNA used as templates
- Note that at the hot start, we have an RNA/DNA hybrid; following will just be dsDNA
Annealing of Primers
Oligonucleotide primers specific for the gene of interest are annealed by rapidly lowering the temperature
Extension of DNA
Heat is increased so that Taq polymerase can make a copy of the single stranded DNA from the primer
How many cycles of qPCR do we go through?
40
What happens at threshold of qPCR?
Enough SYBR green binds to dsDNA that can be detected above noise and is on the exponential part of the curve
Threshold Cycle Value (Ct/Cq)
The point at which fluoresence of SYBR green reach threshold and the exponential part of the curve
Plateau Phase of cPCR
When reaction components are used up, they become rate-limiting and the reaction slows and enters this phase, when the cycles cannot be counted
At what phase of qPCR are measurements taken?
Exponential phase
Purpose of a melt curve
Shows whether or not more than one product was made
How many curves do you expect if there is only one product in a melt curve?
1
What is a melt curve?
A gradual rise in temperature which melts dsDNA and causes an abrupt change in fluorescence
No template control (NTC)
- Only contains master mix
- Should not give a Cq value
- If a Cq value was given, there is extraneous and exogenous DNA (poor lab technique) - just repeat qPCR
No Reverse Transcriptase (NRT)
- Contains master mix and RNA
- Should not give a Cq value
- If a Cq value was given and there is no Cq value from NTC, gDNA from cockroach must have been in the sample
- Run again with DNase
Positive Control
- cDNA that contain qPCR target sequence
- Should give a Cq value
- If doesn’t, qPCR was incorrectly set up, so rerun qPCR
Negative Control
- cDNA that does not contain the qPCR target sequence
- Should not give a qPCR value
- If fails, we are using the wrong primers
- We don’t do this because all DNA has period and actin
What is the reference gene that we used?
B-actin
What assumption do we make with a use of a reference gene?
The independent variable doesn’t affect it
How are we determining levels of period mRNA in groups?
Relative levels using a reference gene to determine that all the samples have the same total cDNA
What do differences of Cq values of beta-actin suggest?
There are different amounts of cDNA added to the sample (not necessarily changes in mRNA)
- If this is the case, Cq values for period will be normalized to Cq values for beta-actin to adjust for any differences
Differences between period and beta-actin qPCR
- Primers
- Annealing temperatures
Change in Cq Method with a reference gene
- Subtract Cqperiod from Cqactin
- 2 to the power of this difference gives the relative expression in terms of arbitrary units
