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Lab Techniques in Neuroscience > module 1 > Flashcards

module 1 Flashcards

1
Q

what is the IV

A

manipulated variable

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2
Q

what is the DV

A

outcome variable

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3
Q

what are we measuring when we measure gene expression

A

how much RNA or protein has been made from the template DNA

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4
Q

what method can be used to measure small amounts of mRNA

A

qPCR

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5
Q

what proteins promote an increase in PER and CRY expression

A

CLOCK and BMAL1

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6
Q

what proteins disrupt CLOCK and BMAL1 activation

A

PER and CRY

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7
Q

what is the master pacemaker

A

suprachiasmatic nucleus in the hypothalamus

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8
Q

what stimulates a wave of cellular activity in the SCN

A

light activating neurons in the retinohypothalamic tract

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9
Q

what is a pilot study

A

small scale, preliminary study run before a larger scale study
- determines overall feasibility, design parameters, possible adverse events, etc

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10
Q

what is falsifiability

A

the criterion that means that you need to be able to show that your hypothesis is false

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11
Q

what is an operationalized hypothesis

A

hypothesis written in terms of the operations and procedures used to test it
-specific, focused, and makes clear predictions

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12
Q

what is internal validity

A

how reliable and replicable the results are and if you can determine the causal relationship between variables

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13
Q

what does having reliable measurements mean

A

give similar results each time they are repeated under the same conditions

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14
Q

what does having replicable results mean

A

experiment is repeated and similar results were obtained

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15
Q

what is external validity

A

how well the research generalizes to other populations/settings

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16
Q

what is ecological validity

A

how well your research mirrors conditions in the natural world

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17
Q

what is predictive validity

A

how well your measures can predict important outcomes

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18
Q

what is a case study

A

thorough analysis relating to a single subject

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19
Q

what do confounding variables influence

A

both the IV and the DV

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20
Q

are all uncontrolled variables confounding variables

A

no

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21
Q

what is a positive control

A

checks that a procedure is working

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22
Q

what is a negative control

A

checks your procedure is not giving you false positive results

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23
Q

what is a vehicle control group

A

used in drug administration studies and controls for other non-drug variables associated with drug administration

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24
Q

what is a sham surgery

A

all variables of the surgery are the same except the step that represents the IV

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25
Q

what does random assignment address

A

addresses the problem that there may be variables that have not been thought of or explicitly controlled for

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26
Q

in addition to randomization of subjects, what should also be randomized

A

order of treatment

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27
Q

what is a within-subjects design

A

each subject experiences all the experimental conditions

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28
Q

what is a matched sample design

A

subjects are matched for that variable between groups

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29
Q

what is attrition

A

loss of subjects before the end of an experiment

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30
Q

what is a quasi experiment

A

when your IV is a subject characteristic (studies involving transgenic (knock in/out) mice) because differences may be due to compensations

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31
Q

what is a single blind study

A

the experimenter knows which group the subject is in but the subject does not

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32
Q

what is a double blind study

A

neither the experimenter nor the subject knows which group the subject is in

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33
Q

what pattern does the log does often show

A

linear response pattern

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34
Q

what is a blocked design in fMRI experiments

A

experimental conditions are alternated, sometimes with a rest period between

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35
Q

what is an event related design in fMRI experiments

A

present test stimuli for a very short period of time with longer time in between for control condition

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36
Q

what does it mean to have convergent evidence

A

multiple experiments using different techniques come to the same conclusion

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37
Q

what is a meta analysis

A

analyzes data from multiple studies on the same topic

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38
Q

what is the difference between a population and a sample

A

population: entire collection of cases of interest
sample: subset of the population

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39
Q

are conclusions of an experiment based on a sample or a population

A

sample from the population

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40
Q

what are descriptive statistics

A

statistics used to describe and visualize the sample data
(mean, SD, etc)

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41
Q

what are inferential statistics

A

statistics used to determine the probability that your samples are from different populations

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42
Q

can you prove that the samples are from different populations with inferential statistics

A

no

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43
Q

what is stratified random sampling

A

sampling where you ensure there is equal representation of groups within a population
(helps generalize to entire population)

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44
Q

what is the null hypothesis

A

no difference between samples (groups)

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45
Q

what is a type I error

A

you reject the null hypothesis and conclude that the groups differ when they do not

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46
Q

what is the alpha value

A

the chance of a type I error (usually up to 5% - p < 0.05)

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47
Q

when is a statistically significant difference between groups obtained

A

if the test statistic meets or exceeds the critical value determined by alpha

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48
Q

what is a type II error

A

you accept the null hypothesis but it is false

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49
Q

what is the probability of making a type II error called

A

beta

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50
Q

what is the power of a test

A

1 - beta
want beta to be as small as possible to give yourself the greatest chance of finding an effect if there is one

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51
Q

what will diminish the chance of making a type II error

A

bigger sample sizes

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52
Q

what is a factorial design

A

experiment with more than one IV

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53
Q

how many IV and levels are there in a 2x2 factorial design

A

2 IV, each with 2 levels

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54
Q

how do you determine the number of groups in a factorial design

A

multiply the numbers together

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55
Q

which axes are the DV and IV on

A

DV on y axis
IV on x axis

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56
Q

to make comparisons between groups in a factorial design, how many variables can differ between them

A

only one

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57
Q

what is a within subjects factorial design

A

each participant receives every level of every IV

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58
Q

what is the beer-lambert law

A

relates the absorption of light to the properties of the material through which the light is traveling

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59
Q

what can we determine based off of the beer-lambert law

A

the concentration of protein in the solution

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60
Q

what was an unethical study that occurred in tuskegee and why was it unethical

A

tuskegee syphilis study
- were not told they had syphilis
- were not asked for consent
- were not offered treatment

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61
Q

what is the common rule policy

A

regulates human research studies under federal regulations

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62
Q

what oversees the IRB

A

office of human research protection (OHRP)

63
Q

what is the belmont report

A

foundation for current ethical guidelines
- respect for persons
- beneficence
- justice

64
Q

what animals does the animal welfare act cover

A

warm-blooded animals but not birds, rats, mice, or farm animals

65
Q

what regulations cover all vertebrate animals

A

PHS policy on humane care and use of laboratory animals
overseen by office of laboratory animal welface

66
Q

what committee must oversee all animal research

A

institutional animal care and use committee (IACUC)

67
Q

what is fraud

A

outright fabrication of data or unjustified conclusions

68
Q

what is bias

A

beliefs that cause data bias if there is any subjectivity in the measure

69
Q

what are three steps to prevent bias

A
  1. analyze all data blind to experimental conditions
  2. double check all data
  3. automate procedures and data handling where possible
70
Q

what are two steps to show research is not fraudulent

A
  1. keep detailed notebooks according to current best practices
  2. maintain files with raw data
71
Q

what is an advantage of qPCR

A

sensitive technique that can detect very low levels of mRNA

72
Q

what is the brain in a cockroach

A

2 fused ganglia

73
Q

where does the ventral nerve cord arise from

A

the “brain”

74
Q

how many ganglia do cockroaches have

A

3 ganglia in thorax
6 ganglia in abdomen

75
Q

what is RNA broken down by

A

RNase
- ribonuclease enzymes that are present in tissues

76
Q

why do we not use organic extraction

A

you need to use a fume hood for organic extraction

77
Q

what happens during organic extraction

A

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

78
Q

what is contained in each of the three phases produced in an organic extraction

A

organic phase and interphase contain denatured proteins, lipids, and gDNA

aqueous phase contains RNA

79
Q

how is the RNA recovered from the aqueous phase in organic extraction

A

alcohol precipitation and rehydration

80
Q

what are the benefits of organic extraction

A

rapid denaturation of nucleases and stabilization of RNA
good method for removing gDNA
scalable technique

81
Q

what are the drawbacks of organic extraction

A

use/disposal of organic reagents
manually intensive processing

81
Q

what happens during the spin column method of RNA isolation

A

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

81
Q

what binds to the membrane in the spin column method

A

RNA and DNA

82
Q

what are the benefits of the spin column method

A

convenient, easy
good for single sample or 96-well processing
can be automated

83
Q

how is DNA removed from the sample in spin column method

A

washed several times and any DNA left is degraded with DNase

84
Q

what are the drawbacks of the spin column method

A

spin columns can easily clog
limited binding capacity
can retain gDNA

85
Q

what is the prep system we will use in lab for RNA extraction

A

ReliaPrep RNA Tissue Miniprep system

86
Q

what kind of buffer is added to our tissue in the spin column method

A

lysis buffer is added to the tissue and then dissected tissue will be homogenized in the lysis buffer

87
Q

what is homogenization

A

mechanical process where the tissue is ground up in a liquid (breakdown at tissue level)

88
Q

what is lysis

A

cell membranes are broken (cells are lysed)

89
Q

what are the two main components of the lysis buffer

A

guanidine thiocyanate
1-thioglycerol

90
Q

what does the guanidine thiocyanate do in the lysis buffer

A

lyses cells
disrupts nucleoprotein complexes
inactivated RNAses

91
Q

what does the 1-thioglycerol do in the lysis buffer

A

inactivated RNAses

92
Q

how is the gDNA sheared after homogenization in the lysis buffer

A

pipetting up and down 7-10 times

93
Q

why did we make cDNA from the RNA

A

we want to measure levels of period mRNA but levels are too low to measure directly so they need to be amplified

94
Q

how can RNA be amplified

A

it cannot be

95
Q

how can DNA be amplified

A

polymerase chain reaction (PCR)

96
Q

what does the cDNA synthesis method make

A

a single cDNA cope of each of the mRNA molecules in the sample

97
Q

what is the ratio of mRNA:cDNA in our experiment

A

1:1 so when we quantify the cDNA in the qPCR it will reflect the quantity of mRNA in the sample

98
Q

for qPCR why do we need to start with the same amount of total nucleic acid for each sample

A

so comparisons between the samples are accurate

99
Q

what happens when RNA is dissolved in water

A

it absorbs light

100
Q

what is the peak absorbance of RNA dissolved in water

A

260nm

101
Q

how is RNA concentration determined

A

its absorbance at 260nm is used to determine concentration using a spectrophotometer and the beer lambert law

102
Q

what is used to determine RNA purity

A

A160:A280

103
Q

what should the A260:A280 ratio for pure RNA in water

A

about 2
because the absorbance at 260nm is twice the absorbance at 280nm

104
Q

what do you need to make cDNA

A

a primer (short length of DNA) to enable the enzyme to start the copying process

105
Q

what is an oligo

A

short length of DNA

106
Q

what primer did we use in our cDNA synthesis

A

oligo(dT)

107
Q

what does the dT part of oligo(dT) mean

A

it is a sequence containing only thymine molecules

108
Q

why did we want to use a primer containing thymine molecules

A

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

109
Q

what are two other types of primers that can be used for cDNA synthesis other than the oligo(dT) primer

A
  • primer specific for a particular target sequence
  • random primer that consists of multiple short primers
110
Q

what are the 6 things needed for cDNA synthesis

A
  1. RNA template (already in tube)
  2. buffer (+DTT+RNase inhibitor)
  3. water
  4. dNTPs
  5. primers
  6. reverse transcriptase
111
Q

what is qPCR used for

A

to both amplify (by PCR) and detect in real time a specific sequence of DNA

112
Q

what is qPCR also known as

A

real-time PCR

113
Q

what is the difference between regular PCR and qPCR

A

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

114
Q

what happens to the amount of DNA with every PCR cycle

A

it doubles until you run out of the ingredients to make DNA

115
Q

how can RNA be measured with qPCR

A

it cannot, needs to be reverse transcribed into DNA first

116
Q

what is RT-PCR

A

reverse transcription PCR
- when the first step in the PCR procedure is to reverse transcribe the RNA into DNA

117
Q

is RNA ever directly amplified

A

no

118
Q

what are two methods of detecting and quantifying amplified DNA products in qPCR

A

TaqMan Probe
SYBR green

119
Q

what is a TaqMan probe

A

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
120
Q

what is the mechanism of SYBR green

A

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

121
Q

what are the 8 things that are in a qPCR reaction tube

A

template
primers (forward and reverse)
nucleotides
buffer
MgCl2
Taq polymerase
SYBR green
water

122
Q

what is the purpose of the first heating step in qPCR

A

hot start Taq polymerase so it is activated

123
Q

what is the enzyme that will made a copy of each DNA strand

A

Taq polymerase

124
Q

what is the first step of qPCR

A

denaturation
-melts the ds helix giving 2 single strands of DNA to use as templates
(our template is initially a DNA-RNA hybrid)

125
Q

what is the second step of qPCR

A

annealing of primers
-temp is rapidly lowered for 10 sec so the primers can bind specifically to each ssDNA template

126
Q

what is the third step of qPCR

A

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

127
Q

when can the SYBR green be detected

A

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

128
Q

what is the CT or Cq

A

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

129
Q

how is a melt curve created

A

temp is gradually raised which melts dsDNA and causes an abrupt change in fluorescence

130
Q

what does a melt curve tell us

A

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

131
Q

what does a no template control include

A

NTC has only master mix (no DNA template)

132
Q

should the NTC give a Cq value

A

no
- if it does it means there is extraneous DNA contamination

133
Q

what does a no reverse transcriptase control include

A

NRT has master mix and RNA

134
Q

should the NRT give a Cq value

A

no
- if it does and NTC does not give Cq value, suggests gDNA contamination of RNA sample

135
Q

what does the positive control in qPCR contain

A

cDNA that contains the qPCR target sequence

136
Q

should the positive control give a Cq value in qPCR

A

yes
-if does not, qPCR was set up incorrectly

137
Q

what does the negative control in qPCR contain

A

cDNA that does not contain the qPCR sequence

138
Q

should the negative control give a Cq value in qPCR

A

no
-if it does, primers are binding to non-target DNA

139
Q

how would you determine the actual levels of a target sequence by qPCR

A

need to run a standard curve with known quantities of the target sequence (we wont do this)

140
Q

to determine the relative levels of period mRNA in each group, what needs to happen with the starting cDNA

A

need to make sure we started with the same total amount of cDNA in each of our qPCR reactions

141
Q

how do we determine if we started with the same amount of cDNA in each reaction

A

use a reference gene

142
Q

what reference gene are we using

A

B-actin

143
Q

should mRNA levels of the reference gene change

A

no, the IV should not change the expression of the reference gene

144
Q

what do differences in Cq values generated in qPCR for B-actin reflect

A

the differences in total amount of cDNA added to each reaction/for each sample (and not changes in mRNA levels)

145
Q

what are the two differences between the qPCR procedure for period and for B-actin

A

different primer pairs will be used
different annealing temps will be used

146
Q

what does the CT/Cq value represent

A

the number of PCR cycles it took to reach the threshold of fluorescence

147
Q

how many peaks should the melt curve have

A

one
- if there are more it means the primers were not specific to the target or they are also binding to gDNA

148
Q

what should happen with your positive control (data analysis)

A

should amplify and give a CT/Cq value

149
Q

what should happen with your NTC (data analysis)

A

should not amplify because there is no template
no CT/Cq value

150
Q

what should happen with your NRT (data analysis)

A

should not amplify if the RNA sample only contains RNA
no CT/Cq value

151
Q
A

Lab Techniques in Neuroscience (3 decks)

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