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
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
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
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
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
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
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
8
Q
full-body knockout mice nomenclature
A
- geneX^-/-
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
10
Q
Cre-loxP nomenclature
A
- geneX^fl/fl or gene-promoter^Cre GeneX^fl/fl
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
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
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
14
Q
DTR system
A
- diptheria toxin receptor system is used to knockout specific cell types
15
Q
DTR
A
- diptheria toxin is a potent toxin which requires DTR on cell surfaces to work
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
17
Q
in vitro
A
- the study of microorganisms, cellular responses, and other molecules outside of their normal biological context
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
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
20
Q
reporter gene: GFP
A
- can be physically linked to proteins of interest to track their cellular location
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
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
23
Q
how is the magnitude of siRNA transfection measured
A
- must use western blot or ELISA
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
25
siRNA transfection: negative control
- random, non-specific RNA should be used to see if nucleic acid can alter the behaviour of the cell
26
immunofluorescent (IF) microscopy (2)
- used to determine cellular localization of proteins
- limited to ~four different fluorochromes in one sample
27
what are the steps for IF microscopy (3)
- 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
28
IF microscopy: positive control
- use any tissue or cell that is known to express the protein of interest in abundance
29
IF microscopy: negative control
- use any tissue or cell that is known to not express the protein of interest/target antigen
30
ELISA
- enzyme-linked immunosorbant assay is used to accurately quantify a protein of interest
31
ELISA: direct method (5)
- 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
32
ELISA: sandwich method (5)
- 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
33
ELISA: advantage
- highly specific due to dual antibody detection
34
ELISA: disadvantage
- no ability to measure size of protein
35
ELISA: postive control
- use a purified protein or peptide known to detected by antibody used
36
ELISA: negative control
- use cells not expressing protein of interest; expect to see no coloured product in result
37
co-immunoprecipitation (co-IP)
- used to determine protein-protein interactions
- gold standard for detecting protein interactions, and especially powerful when paired with sequencing
38
co-IP steps (4)
- 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
co-IP: negative control
- use proteins not related to the target protein; antibodies should not bind at all and no protein will be detected
40
SDS-PAGE
- simple way to examine proteins in sample by relative size
41
SDS-PAGE steps (4)
- 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
SDS-PAGE characteristics (3)
- may need to induce protein expression in cells
- can detect proteins, but difficult to quantify
- staining is non-specific
43
SDS-PAGE: negative control
- use non-induced cells which have essentially have no proteins run through PAGE; expect to see no protein expression
44
western blot
- identify specific proteins from a complex mixture of proteins extracted from cells
45
western blot steps (6)
- 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
western blot: positive control
- co-stain for a constitutively expressed protein to normalize amount of protein loaded
47
western blot: negative control
- co-stain for a protein that is not expressed
48
flow cytometry
- powerful cell phenotyping tool that detects relative number of proteins and allows for estimation of cell proportion and population
49
FACS
- same as flow cytometry, but individual cell types are isolated through fluorescence (fluorescence activated cell-sorting)
50
flow cytometry/FACS: steps (4)
- 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
interpretation of flow cytometry/FACS results (3)
- 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
flow cytometry/FACS: gating
- sequential identification and refinement of a cellular population of interest using panel of molecules
53
characteristics of flow cytometry/FACS
- 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
flow cytometry/FACS: controls
- beyond the scope of our course, but ensure to state that appropriate controls are used
55
mass cytometry (cyTOF) (3)
- 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
mass cytometry analysis (2)
- done using time-of-flight mass spectrometer (TOF)
- major hurdle, complex algorithms to handle amount of data
57
quantitative PCR (qPCR) (2)
- 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
qPCR steps (4)
- 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
qPCR: threshold cycle(2)
- 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
qPCR: postitive control
- use DNA of predetermined value that is known to express the target; should produce a standardized curve
61
qPCR: negative control
- perform the experiment with no amplification
62
reverse transcriptase qPCR
- can be used to quantify gene expression
63
RT qPCR steps (4)
- 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
RT qPCR: advantages (3)
- quick, easy, and specific
- easy to quantify unculturable organisms
- easy way to quantify gene expression
65
RT qPCR disadvantages (2)
- mRNA expression does not equal protein expression
- best to confirm results with additional experiment, such as western blot
66
RT qPCR: positive control
- use housekeeping mRNA of a pre-determined value
67
RT qPCR: negative control
- perform experiment with all conditions equal except for the exclusion of the reverse transcriptase; tests for contaminating DNA
68
single cell RNA sequencing (scRNA-Seq) (3)
- 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
Sangers DNA Sequencing
- used to sequence unknown genes
70
Sangers DNA Sequencing: steps (5)
- 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
Sangers DNA Sequencing: advantages
- extremely accurate
72
Sangers DNA Sequencing: disadvantages (2)
- very low throughput
- works best with prior knowledge of template (reference genome)
73
next-gen sequencing
- function
- comparison to Sanger (2)
- 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
loss of function: methods to remove component from system (3)
- gene knockouts
- study natural mutations in an organism
- germ-free mice
75
loss of function: methods to decrease amount or block function in some way (4)
- 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
gain of function: methods to add something system is lacking or increase concentration of limiting component (2)
- adding purified component to system
- introduce a transgene (over-expression of protein constitutive expression
77
gain of function: methods to increase amount of active component in system (2)
- 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
what is a good control for western blots
- beta-actin; constitutively expressed in all eukaryotic cells and can be used to confirm protein loading is the same across gel
79
flow cytometry vs CyTOF (2)
- 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
CyTOF and dimenstionality
- computationally demanding as plots need to be examined at extremely high dimensions that the human mind cannot comprehend
81
CyTOF markers and plots
- for n amount of parameter/markers, we have to access 2^n plots
82
scRNAseq vs CyTOF (2)
- 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
DROP-seq
- single cell RNA sequencing using microfluidics and barcoded beads
84
cite-seq
- a method for performing RNA sequencing along with gaining quantitative and qualitative information on surface proteins with available antibodies on a single cell level
