module 1 Flashcards
what is the IV
manipulated variable
what is the DV
outcome variable
what are we measuring when we measure gene expression
how much RNA or protein has been made from the template DNA
what method can be used to measure small amounts of mRNA
qPCR
what proteins promote an increase in PER and CRY expression
CLOCK and BMAL1
what proteins disrupt CLOCK and BMAL1 activation
PER and CRY
what is the master pacemaker
suprachiasmatic nucleus in the hypothalamus
what stimulates a wave of cellular activity in the SCN
light activating neurons in the retinohypothalamic tract
what is a pilot study
small scale, preliminary study run before a larger scale study
- determines overall feasibility, design parameters, possible adverse events, etc
what is falsifiability
the criterion that means that you need to be able to show that your hypothesis is false
what is an operationalized hypothesis
hypothesis written in terms of the operations and procedures used to test it
-specific, focused, and makes clear predictions
what is internal validity
how reliable and replicable the results are and if you can determine the causal relationship between variables
what does having reliable measurements mean
give similar results each time they are repeated under the same conditions
what does having replicable results mean
experiment is repeated and similar results were obtained
what is external validity
how well the research generalizes to other populations/settings
what is ecological validity
how well your research mirrors conditions in the natural world
what is predictive validity
how well your measures can predict important outcomes
what is a case study
thorough analysis relating to a single subject
what do confounding variables influence
both the IV and the DV
are all uncontrolled variables confounding variables
no
what is a positive control
checks that a procedure is working
what is a negative control
checks your procedure is not giving you false positive results
what is a vehicle control group
used in drug administration studies and controls for other non-drug variables associated with drug administration
what is a sham surgery
all variables of the surgery are the same except the step that represents the IV
what does random assignment address
addresses the problem that there may be variables that have not been thought of or explicitly controlled for
in addition to randomization of subjects, what should also be randomized
order of treatment
what is a within-subjects design
each subject experiences all the experimental conditions
what is a matched sample design
subjects are matched for that variable between groups
what is attrition
loss of subjects before the end of an experiment
what is a quasi experiment
when your IV is a subject characteristic (studies involving transgenic (knock in/out) mice) because differences may be due to compensations
what is a single blind study
the experimenter knows which group the subject is in but the subject does not
what is a double blind study
neither the experimenter nor the subject knows which group the subject is in
what pattern does the log does often show
linear response pattern
what is a blocked design in fMRI experiments
experimental conditions are alternated, sometimes with a rest period between
what is an event related design in fMRI experiments
present test stimuli for a very short period of time with longer time in between for control condition
what does it mean to have convergent evidence
multiple experiments using different techniques come to the same conclusion
what is a meta analysis
analyzes data from multiple studies on the same topic
what is the difference between a population and a sample
population: entire collection of cases of interest
sample: subset of the population
are conclusions of an experiment based on a sample or a population
sample from the population
what are descriptive statistics
statistics used to describe and visualize the sample data
(mean, SD, etc)
what are inferential statistics
statistics used to determine the probability that your samples are from different populations
can you prove that the samples are from different populations with inferential statistics
no
what is stratified random sampling
sampling where you ensure there is equal representation of groups within a population
(helps generalize to entire population)
what is the null hypothesis
no difference between samples (groups)
what is a type I error
you reject the null hypothesis and conclude that the groups differ when they do not
what is the alpha value
the chance of a type I error (usually up to 5% - p < 0.05)
when is a statistically significant difference between groups obtained
if the test statistic meets or exceeds the critical value determined by alpha
what is a type II error
you accept the null hypothesis but it is false
what is the probability of making a type II error called
beta
what is the power of a test
1 - beta
want beta to be as small as possible to give yourself the greatest chance of finding an effect if there is one
what will diminish the chance of making a type II error
bigger sample sizes
what is a factorial design
experiment with more than one IV
how many IV and levels are there in a 2x2 factorial design
2 IV, each with 2 levels
how do you determine the number of groups in a factorial design
multiply the numbers together
which axes are the DV and IV on
DV on y axis
IV on x axis
to make comparisons between groups in a factorial design, how many variables can differ between them
only one
what is a within subjects factorial design
each participant receives every level of every IV
what is the beer-lambert law
relates the absorption of light to the properties of the material through which the light is traveling
what can we determine based off of the beer-lambert law
the concentration of protein in the solution
what was an unethical study that occurred in tuskegee and why was it unethical
tuskegee syphilis study
- were not told they had syphilis
- were not asked for consent
- were not offered treatment
what is the common rule policy
regulates human research studies under federal regulations
what oversees the IRB
office of human research protection (OHRP)
what is the belmont report
foundation for current ethical guidelines
- respect for persons
- beneficence
- justice
what animals does the animal welfare act cover
warm-blooded animals but not birds, rats, mice, or farm animals
what regulations cover all vertebrate animals
PHS policy on humane care and use of laboratory animals
overseen by office of laboratory animal welface
what committee must oversee all animal research
institutional animal care and use committee (IACUC)
what is fraud
outright fabrication of data or unjustified conclusions
what is bias
beliefs that cause data bias if there is any subjectivity in the measure
what are three steps to prevent bias
- analyze all data blind to experimental conditions
- double check all data
- automate procedures and data handling where possible
what are two steps to show research is not fraudulent
- keep detailed notebooks according to current best practices
- maintain files with raw data
what is an advantage of qPCR
sensitive technique that can detect very low levels of mRNA
what is the brain in a cockroach
2 fused ganglia
where does the ventral nerve cord arise from
the “brain”
how many ganglia do cockroaches have
3 ganglia in thorax
6 ganglia in abdomen
what is RNA broken down by
RNase
- ribonuclease enzymes that are present in tissues
why do we not use organic extraction
you need to use a fume hood for organic extraction
what happens during organic extraction
sample is homogenized in a phenol-based solution and then centrifuged
- when centrifuged it separates into a lower organic phase, a middle interphase, and an upper aqueous phase
what is contained in each of the three phases produced in an organic extraction
organic phase and interphase contain denatured proteins, lipids, and gDNA
aqueous phase contains RNA
how is the RNA recovered from the aqueous phase in organic extraction
alcohol precipitation and rehydration
what are the benefits of organic extraction
rapid denaturation of nucleases and stabilization of RNA
good method for removing gDNA
scalable technique
what are the drawbacks of organic extraction
use/disposal of organic reagents
manually intensive processing
what happens during the spin column method of RNA isolation
filter-based, spin columns use membranes across the bottom of a plastic column
- tissue samples are homogenized in a lysis buffer, added to column, and centrifuged
what binds to the membrane in the spin column method
RNA and DNA
what are the benefits of the spin column method
convenient, easy
good for single sample or 96-well processing
can be automated
how is DNA removed from the sample in spin column method
washed several times and any DNA left is degraded with DNase
what are the drawbacks of the spin column method
spin columns can easily clog
limited binding capacity
can retain gDNA
what is the prep system we will use in lab for RNA extraction
ReliaPrep RNA Tissue Miniprep system
what kind of buffer is added to our tissue in the spin column method
lysis buffer is added to the tissue and then dissected tissue will be homogenized in the lysis buffer
what is homogenization
mechanical process where the tissue is ground up in a liquid (breakdown at tissue level)
what is lysis
cell membranes are broken (cells are lysed)
what are the two main components of the lysis buffer
guanidine thiocyanate
1-thioglycerol
what does the guanidine thiocyanate do in the lysis buffer
lyses cells
disrupts nucleoprotein complexes
inactivated RNAses
what does the 1-thioglycerol do in the lysis buffer
inactivated RNAses
how is the gDNA sheared after homogenization in the lysis buffer
pipetting up and down 7-10 times
why did we make cDNA from the RNA
we want to measure levels of period mRNA but levels are too low to measure directly so they need to be amplified
how can RNA be amplified
it cannot be
how can DNA be amplified
polymerase chain reaction (PCR)
what does the cDNA synthesis method make
a single cDNA cope of each of the mRNA molecules in the sample
what is the ratio of mRNA:cDNA in our experiment
1:1 so when we quantify the cDNA in the qPCR it will reflect the quantity of mRNA in the sample
for qPCR why do we need to start with the same amount of total nucleic acid for each sample
so comparisons between the samples are accurate
what happens when RNA is dissolved in water
it absorbs light
what is the peak absorbance of RNA dissolved in water
260nm
how is RNA concentration determined
its absorbance at 260nm is used to determine concentration using a spectrophotometer and the beer lambert law
what is used to determine RNA purity
A160:A280
what should the A260:A280 ratio for pure RNA in water
about 2
because the absorbance at 260nm is twice the absorbance at 280nm
what do you need to make cDNA
a primer (short length of DNA) to enable the enzyme to start the copying process
what is an oligo
short length of DNA
what primer did we use in our cDNA synthesis
oligo(dT)
what does the dT part of oligo(dT) mean
it is a sequence containing only thymine molecules
why did we want to use a primer containing thymine molecules
it will bind to adenine
each mRNA has a polyA tail so the oligo(dT) primer will selectively bind to mRNA because only mRNA has a polyA tail
what are two other types of primers that can be used for cDNA synthesis other than the oligo(dT) primer
- primer specific for a particular target sequence
- random primer that consists of multiple short primers
what are the 6 things needed for cDNA synthesis
- RNA template (already in tube)
- buffer (+DTT+RNase inhibitor)
- water
- dNTPs
- primers
- reverse transcriptase
what is qPCR used for
to both amplify (by PCR) and detect in real time a specific sequence of DNA
what is qPCR also known as
real-time PCR
what is the difference between regular PCR and qPCR
when the DNA is detected
- in regular PCR the accumulation of DNA is measured at the end of the procedure
- in qPCR, the DNA is measured during/throughout the procedure
what happens to the amount of DNA with every PCR cycle
it doubles until you run out of the ingredients to make DNA
how can RNA be measured with qPCR
it cannot, needs to be reverse transcribed into DNA first
what is RT-PCR
reverse transcription PCR
- when the first step in the PCR procedure is to reverse transcribe the RNA into DNA
is RNA ever directly amplified
no
what are two methods of detecting and quantifying amplified DNA products in qPCR
TaqMan Probe
SYBR green
what is a TaqMan probe
a sequence-specific probe that can be labeled with a reporter and quencher
- the quencher prevents detection of the reporter when they are close together
- the Taq DNA pol will degrade sequences in its path via 5’-exonuclease activity which will release the reporter to be detected
what is the mechanism of SYBR green
binds to the minor groove in dsDNA and emits a green light when excited by blue light
- only in dsDNA
- not specific for a specific dsDNA sequence
what are the 8 things that are in a qPCR reaction tube
template
primers (forward and reverse)
nucleotides
buffer
MgCl2
Taq polymerase
SYBR green
water
what is the purpose of the first heating step in qPCR
hot start Taq polymerase so it is activated
what is the enzyme that will made a copy of each DNA strand
Taq polymerase
what is the first step of qPCR
denaturation
-melts the ds helix giving 2 single strands of DNA to use as templates
(our template is initially a DNA-RNA hybrid)
what is the second step of qPCR
annealing of primers
-temp is rapidly lowered for 10 sec so the primers can bind specifically to each ssDNA template
what is the third step of qPCR
extension of DNA
- temp is raised so Taq polymerase can made copy of each DNA strand
SYBR green binds to any dsDNA and provided fluorescent signal
when can the SYBR green be detected
after many cycles, the amount of SYBR green bound to the dsDNA reaches the threshold at which the fluorescence can be detected above the background and is on the linear/exponential part of the curve
what is the CT or Cq
CT: threshold cycle value
Cq: quantification cycle value
point at which the fluorescence can be detected above the background and is on the linear/exponential part of the curve
how is a melt curve created
temp is gradually raised which melts dsDNA and causes an abrupt change in fluorescence
what does a melt curve tell us
information about the products that have been produced
- if there is more than one peak, it indicates more than one product was made and the reaction was not specific
what does a no template control include
NTC has only master mix (no DNA template)
should the NTC give a Cq value
no
- if it does it means there is extraneous DNA contamination
what does a no reverse transcriptase control include
NRT has master mix and RNA
should the NRT give a Cq value
no
- if it does and NTC does not give Cq value, suggests gDNA contamination of RNA sample
what does the positive control in qPCR contain
cDNA that contains the qPCR target sequence
should the positive control give a Cq value in qPCR
yes
-if does not, qPCR was set up incorrectly
what does the negative control in qPCR contain
cDNA that does not contain the qPCR sequence
should the negative control give a Cq value in qPCR
no
-if it does, primers are binding to non-target DNA
how would you determine the actual levels of a target sequence by qPCR
need to run a standard curve with known quantities of the target sequence (we wont do this)
to determine the relative levels of period mRNA in each group, what needs to happen with the starting cDNA
need to make sure we started with the same total amount of cDNA in each of our qPCR reactions
how do we determine if we started with the same amount of cDNA in each reaction
use a reference gene
what reference gene are we using
B-actin
should mRNA levels of the reference gene change
no, the IV should not change the expression of the reference gene
what do differences in Cq values generated in qPCR for B-actin reflect
the differences in total amount of cDNA added to each reaction/for each sample (and not changes in mRNA levels)
what are the two differences between the qPCR procedure for period and for B-actin
different primer pairs will be used
different annealing temps will be used
what does the CT/Cq value represent
the number of PCR cycles it took to reach the threshold of fluorescence
how many peaks should the melt curve have
one
- if there are more it means the primers were not specific to the target or they are also binding to gDNA
what should happen with your positive control (data analysis)
should amplify and give a CT/Cq value
what should happen with your NTC (data analysis)
should not amplify because there is no template
no CT/Cq value
what should happen with your NRT (data analysis)
should not amplify if the RNA sample only contains RNA
no CT/Cq value
