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MICB 402 > Methods > Flashcards

Methods Flashcards

1
Q

positive control (2)

A
  • group that receives treatment with a known results
  • indicate that the procedure is optimized and working correctly
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2
Q

negative control (2)

A
  • group that is treated the same as all other groups, but is expected to show no change during the treatment
  • indicate whether the procedure is creating false positives
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3
Q

gain of function (2)

A
  • genetically altering an organism to have some enhanced function
  • used to test if something is sufficient to trigger a phenotype
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4
Q

it is thought that virus A is infecting humans because it binds to surface receptor A, how could we test our hypothesis with a gain of function approach?

A
  • alter mice cells to express surface receptor A and see if virus A is able to infect the mouse
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5
Q

loss of function (2)

A
  • genetically alter organism to lack a function that the wild-type normally has
  • used to test if something is necessary for a phenotype to occur
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6
Q

it is thought that virus A is infecting humans because it binds to surface receptor A, how could we test our hypothesis with a loss of function approach?

A
  • alter human cells to lose expression of surface receptor A and see if virus A is still able to infect these cells
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7
Q

full-body knockout mice (3)

A
  • completely deficient for targeted gene
  • genes have may roles, so it is difficult to observe the cause of the resulting phenotype
  • knockouts can be embryologically lethal
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8
Q

full-body knockout mice nomenclature

A
  • geneX^-/-
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9
Q

Cre-loxP system steps (2)

A
  • gene to be targeted out is flanked with palindromic loxP sequences
  • cre recombinase activity will remove sequences flanked by loxP
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10
Q

Cre-loxP nomenclature

A
  • geneX^fl/fl or gene-promoter^Cre GeneX^fl/fl
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11
Q

how can Cre-loxP be used to make knockouts specific to certain cell types (2)

A
  • insert Cre into genomes downstream of a specific cell marker promoter
  • only cells that express that specific marker will also express Cre in their cells
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12
Q

how can Cre-loxP be used to induce specific expression of other genes

A
  • insert floxed stop codon downstream of a promoter, but upstream of the coding sequence so that the gene will only be expressed when Cre activity remove the stop codon
  • Cre reporter strain used to assess specificity of Cre activity
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13
Q

Cre-ER system (3)

A
  • temporal control of gene knockouts
  • attachment of a modified estrogen receptor (ER) to Cre allows use to use drug to induce Cre activity
  • Cre-ER is localized in the cytoplasm and can’t access loxP sites
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14
Q

DTR system

A
  • diptheria toxin receptor system is used to knockout specific cell types
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15
Q

DTR

A
  • diptheria toxin is a potent toxin which requires DTR on cell surfaces to work
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16
Q

how does the DTR system work in mice (2)

A
  • wildtype mice do not express DTR, so DT has no effect on them
  • we can introduce the DTR gene in specific cell types ad then knock these cell types out by administering DT
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17
Q

in vitro

A
  • the study of microorganisms, cellular responses, and other molecules outside of their normal biological context
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18
Q

which common techniques are done in vitro (8)

A
  • reporter gene assays
  • IF microscopy
  • ELISA
  • SDS-PAGE
  • Western Blot
  • Immunoprecipitation
  • FACS
  • scRNA-Seq
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19
Q

reporter genes (2)

A
  • reveal when a gene is being expressed
  • reporter gene sequences can be inserted downstream of the gene you want to measure expression of
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20
Q

reporter gene: GFP

A
  • can be physically linked to proteins of interest to track their cellular location
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21
Q

small interfering (si)RNA transfection
- what it is
- process (3)

A
  • siRNA reflects a LOF approach; mRNA is transcribed from the DNA, but it cannot be translated into a protein
  • siRNA is a dsRNA complementary to a target mRNA
  • dicer protein complex processes siRNA into a ssRNA guide, which is then bound by RISC (RNA induced silencing complex)
  • recognition of complementary mRNA by guide RNA results in RISC cleavage of mRNA, preventing translation of targeted mRNA
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22
Q

characteristics of siRNA transfection (4)

A
  • fast and simple to accomplish
  • extremely specific knockdown of target
  • not a total knockout, but useful for examining dose effects
  • very difficult to do in vivo
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23
Q

how is the magnitude of siRNA transfection measured

A
  • must use western blot or ELISA
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24
Q

siRNA transfection: postitive control (2)

A
  • un-transfected cells should be used to see if transfection influences cell behaviour
  • siRNAs known to achieve high levels of knockdown, typically for a constitutively expressed protein
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25
Q

siRNA transfection: negative control

A
  • random, non-specific RNA should be used to see if nucleic acid can alter the behaviour of the cell
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26
Q

immunofluorescent (IF) microscopy (2)

A
  • used to determine cellular localization of proteins
  • limited to ~four different fluorochromes in one sample
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27
Q

what are the steps for IF microscopy (3)

A
  • permeabilize cells with gentle detergent
  • treat with fluorochrome-labelled antibodies specific to protein of interest
  • visualize under a microscope with laser excitation under sufficient resolution
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28
Q

IF microscopy: positive control

A
  • use any tissue or cell that is known to express the protein of interest in abundance
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29
Q

IF microscopy: negative control

A
  • use any tissue or cell that is known to not express the protein of interest/target antigen
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30
Q

ELISA

A
  • enzyme-linked immunosorbant assay is used to accurately quantify a protein of interest
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31
Q

ELISA: direct method (5)

A
  • coat a plate with a sample
  • rinse with primary antibody specific for the protein of interest
  • rinse with a secondary antibody specific for the primary antibody
  • secondary antibody is conjugated to an enzyme which converts substrate into coloured product
  • amount of coloured produced is related to the amount of protein of interest in the sample
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32
Q

ELISA: sandwich method (5)

A
  • coat plate with capture antibody specific for protein of interest
  • rinse with sample
  • rinse with detection antibody also specific to protein of interest (at a different epitope)
  • second antibody is conjugated to an enzyme which converts substrate into coloured product
  • amount of coloured produced is related to the amount of protein of interest in the sample
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33
Q

ELISA: advantage

A
  • highly specific due to dual antibody detection
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34
Q

ELISA: disadvantage

A
  • no ability to measure size of protein
35
Q

ELISA: postive control

A
  • use a purified protein or peptide known to detected by antibody used
36
Q

ELISA: negative control

A
  • use cells not expressing protein of interest; expect to see no coloured product in result
37
Q

co-immunoprecipitation (co-IP)

A
  • used to determine protein-protein interactions
  • gold standard for detecting protein interactions, and especially powerful when paired with sequencing
38
Q

co-IP steps (4)

A
  • label cells with radioisotope that attaches to protein of interest
  • lyse cells, wash and add antibodies specific to protein of interest (bind to isotope); this will purify the protein and anything bound to it from the rest of the cell
  • disrupt protein-protein bonds with detergent and identify the proteins
  • sequencing of the separated proteins can be done to characterize the interacting proteins
39
Q

co-IP: negative control

A
  • use proteins not related to the target protein; antibodies should not bind at all and no protein will be detected
40
Q

SDS-PAGE

A
  • simple way to examine proteins in sample by relative size
41
Q

SDS-PAGE steps (4)

A
  • SDS denatures proteins and gives them a negative charge
  • SDS-treated proteins can be separated by run through PAGE
  • negatively charged proteins move through the gel at different speeds depending on their size
  • stain gel blue to visualize the proteins
42
Q

SDS-PAGE characteristics (3)

A
  • may need to induce protein expression in cells
  • can detect proteins, but difficult to quantify
  • staining is non-specific
43
Q

SDS-PAGE: negative control

A
  • use non-induced cells which have essentially have no proteins run through PAGE; expect to see no protein expression
44
Q

western blot

A
  • identify specific proteins from a complex mixture of proteins extracted from cells
45
Q

western blot steps (6)

A
  • load and separate protein samples on SDS-PAGE gel
  • electrophoretically transfer fractionated proteins onto a membrane
  • block membrane with neutral protein
  • incubate membrane with primary antibody specific to target protein, wash
  • incubate membrane with labelled secondary antibody specific to the primary antibody; wash
  • incubate blot with substrate and expose the film
46
Q

western blot: positive control

A
  • co-stain for a constitutively expressed protein to normalize amount of protein loaded
47
Q

western blot: negative control

A
  • co-stain for a protein that is not expressed
48
Q

flow cytometry

A
  • powerful cell phenotyping tool that detects relative number of proteins and allows for estimation of cell proportion and population
49
Q

FACS

A
  • same as flow cytometry, but individual cell types are isolated through fluorescence (fluorescence activated cell-sorting)
50
Q

flow cytometry/FACS: steps (4)

A
  • stain cells with fluorochrome-conjugated antibodies specific to proteins of interest; internal proteins can be stained if cells are fixed and permeabilized
  • run samples through cytometer to analyse thousands of cells
  • microfluidic system aligns cells for run past laser to detect different fluorochromes
  • intensity of fluorescence detects relative number of proteins and allows estimation of cell proportion in population
51
Q

interpretation of flow cytometry/FACS results (3)

A
  • forward scatter (horizontal) of laser indicative of cell size
  • size scatter (vertical) of laser indicative of cell complexity
  • results plotted as dot-plan on biaxial graph and analyzed for estimating proportion of population consisting of certain cell type
52
Q

flow cytometry/FACS: gating

A
  • sequential identification and refinement of a cellular population of interest using panel of molecules
53
Q

characteristics of flow cytometry/FACS

A
  • simple to phenotype and quantify many different cell types and can analyze rare cells in a complex population
  • usually limited to ~12 fluorochromes, though new methods are evolving
54
Q

flow cytometry/FACS: controls

A
  • beyond the scope of our course, but ensure to state that appropriate controls are used
55
Q

mass cytometry (cyTOF) (3)

A
  • allows for simultaneous analysis of many more targets
  • same as flow cytometry, but uses antibodies conjugated to rare isotopes instead of fluorochromes
  • allows for measurement of 80+ targets
56
Q

mass cytometry analysis (2)

A
  • done using time-of-flight mass spectrometer (TOF)
  • major hurdle, complex algorithms to handle amount of data
57
Q

quantitative PCR (qPCR) (2)

A
  • use to characterize and quantify nucleic acids
  • can be used to quantify organisms, viruses, and gene expression; eg. amount of pathogenic bacteria that is in specific strain of mouse’s poop
58
Q

qPCR steps (4)

A
  • design primers specific to target of interest
  • perform PCR reaction containing fluorochrome that fluoresces when bound to DNA
  • amount of fluorescence is related to amount of DNA created by PCR and fluorescence is measured every PCR cycle
  • speed that PCR creates DNA depends on how much target sequence there was to begin with; more fluorescence means there was more beginning DNA
59
Q

qPCR: threshold cycle(2)

A
  • cycle where fluorescence of sample increases past a given threshold
  • comparison of this cycle to set of standards will determine the amount of template in starting sample
60
Q

qPCR: postitive control

A
  • use DNA of predetermined value that is known to express the target; should produce a standardized curve
61
Q

qPCR: negative control

A
  • perform the experiment with no amplification
62
Q

reverse transcriptase qPCR

A
  • can be used to quantify gene expression
63
Q

RT qPCR steps (4)

A
  • isolate mRNA from sample
  • use reverse transcriptase to convert mRNA to cDNA
  • use qPCR to quantify cDNA
  • compare expression of gene z to stably expressed housekeeping gene (positive control)
64
Q

RT qPCR: advantages (3)

A
  • quick, easy, and specific
  • easy to quantify unculturable organisms
  • easy way to quantify gene expression
65
Q

RT qPCR disadvantages (2)

A
  • mRNA expression does not equal protein expression
  • best to confirm results with additional experiment, such as western blot
66
Q

RT qPCR: positive control

A
  • use housekeeping mRNA of a pre-determined value
67
Q

RT qPCR: negative control

A
  • perform experiment with all conditions equal except for the exclusion of the reverse transcriptase; tests for contaminating DNA
68
Q

single cell RNA sequencing (scRNA-Seq) (3)

A
  • enables high-throughput analysis of gene expression in tissues
  • isolate single cell from tissues using FACS to analyze expression of different genes in different cell types
  • lower throughput than bulk RNA-Seq, but allows for identification of rare and complex cell types
69
Q

Sangers DNA Sequencing

A
  • used to sequence unknown genes
70
Q

Sangers DNA Sequencing: steps (5)

A
  • PCR is run with primers targeting known sequence
  • dNTPs are mixed with ddNTP (incorporation terminates PCR)
  • each ddNTP is labelled with a unique colour
  • run product through gel capillary for size separation
  • laser excitation and detection by sequencing machine
71
Q

Sangers DNA Sequencing: advantages

A
  • extremely accurate
72
Q

Sangers DNA Sequencing: disadvantages (2)

A
  • very low throughput
  • works best with prior knowledge of template (reference genome)
73
Q

next-gen sequencing
- function
- comparison to Sanger (2)

A
  • high throughput method for sequencing
  • like Sanger, but performed thousands of times in parallel, making it possible to sequence entire genomes/communities
  • no prior knowledge of sequence needed, but less accurate than Sanger
74
Q

loss of function: methods to remove component from system (3)

A
  • gene knockouts
  • study natural mutations in an organism
  • germ-free mice
75
Q

loss of function: methods to decrease amount or block function in some way (4)

A
  • express mutant forms of protein that are non-functional, but also inhibit the function of endogenous protein
  • deplete component from a reaction (use antibodies)
  • gene knockdown (RNA interference, siRNA screens)
  • drugs that target activity
76
Q

gain of function: methods to add something system is lacking or increase concentration of limiting component (2)

A
  • adding purified component to system
  • introduce a transgene (over-expression of protein constitutive expression
77
Q

gain of function: methods to increase amount of active component in system (2)

A
  • removal of negative regulatory control by making protein form that is not subject to inhibition
  • alter regulatory elements in promoters or transcriptional repressor binding sites
78
Q

what is a good control for western blots

A
  • beta-actin; constitutively expressed in all eukaryotic cells and can be used to confirm protein loading is the same across gel
79
Q

flow cytometry vs CyTOF (2)

A
  • flow cytometry: must know markers and make assumptions, making it more of a reductionist approach; only able to identify ~10 proteins
  • CyTOF: more of a systems approach and can sort for ~40 proteins in one panel
80
Q

CyTOF and dimenstionality

A
  • computationally demanding as plots need to be examined at extremely high dimensions that the human mind cannot comprehend
81
Q

CyTOF markers and plots

A
  • for n amount of parameter/markers, we have to access 2^n plots
82
Q

scRNAseq vs CyTOF (2)

A
  • scRNAseq: quantifies mRNA profiles in single cells to determine cell identity, but no protein info
  • CyTOF: quantifies protein profiles in single cells to determine cell identity, but no mRNA info
83
Q

DROP-seq

A
  • single cell RNA sequencing using microfluidics and barcoded beads
84
Q

cite-seq

A
  • a method for performing RNA sequencing along with gaining quantitative and qualitative information on surface proteins with available antibodies on a single cell level

MICB 402 (11 decks)

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