Final review-Methods Flashcards
Flow Cytometry
used to measure size, shape, complexities of cells
-can observe surface protein/glycoprotein expression
Adv:
-accurate cell sorting, subpop analysis
Dis:
-too slow, commonly unnecessary information for single experiment
GFP
Used to tag specific proteins
Adv:
- analyze live organisms
- study viral infections (pre and post infection)
- can study across multiple time points
Dis:
-fluorescent protein may interfere with biology
Southern blot
Uses probe (difference to PCR) -determine DNA fragment size
Elisa
Measure concentration of antibody OR antigen
Assay diagram:
measuring antibody:
-antigen-antibody of interest-labeled antibody
measuring antigen:
-antibody-antigen of interest-antibody-labeled antibody
ADV:
-highly sensitive and specific
DIS:
-labeling can effect immunoreactivity of antibody
Northern blot
Measure RNA levels
microarray
Measure RNA levels
In-situ hybridization
determine RNA or DNA presence in tissue
knock-out
Removing a gene
knock-down
silencing a gene at mRNA level (study function of gene expression without removing it)
- easy gene rescue
- gene not completely removed
Animal models
used in correlation & causation studies
Adv:
- use KO studies with controls possible
- can use clonally identical animals
Dis:
- some ethical concerns
- still only models (not humans)
Human models
Used usually in correlational studies
Adv:
-best representation of human biology
Dis:
- control studies not always possible
- lethal studies not possible
- ethical concerns
gene trapping
randomly mutating genes to try to find gene of interest
ADV:
-wide approach (used when unsure where to start)
DIS:
-non-specific
microarray
removing mRNA to determine expression
–>used to determine what mRNA are expressed in sample
ADV:
-massive parallel sequencing
Humanized animal models
inject animal with human cells-mimics physiology of disease in humans
cell-line
- cells replicate indefinitely
- used for long experiments (over 2 weeks)
- study cancer
CRISPR/CAS9
Cuts a gene at a specific location and can insert another-effects DNA (transcription)
-create loss of function OR gain of function mutations
ADV:
-specifically targeted locations on DNA
DIS:
-possible unwanted modifications due to host repair system
shRNA
creates hairpin at gene of choice-effects RNA (translation
organoids
- observe cell-cell interactions
- observe viral infections in organ systems
cultures with 2 cell types
can mimic more of physiology of disease in humans compared to one cell type cultures
-reflects real cell interactions
- study cancer
- study tissue invasion
gene rescue
Method to see if WT phenotype is restored
Dis: assumes gene was removed and then re-introduced
Western Blot
Used to detect protein and divide them by size
(denatures the proteins)
-measure antigen concentrations
ASSAY diagram:
- smallest
- medium sized
Elispot
Measure amount of antigen or antibody
-monitor cell immune responses
ASSAY diagram:
- all 3
- largest measures antibody
- other 2 measures antigen
ADV:
-sensitive, allows for detection of single cell
DIS:
-cannot quantify amount of protein secreted by cell
immunohistochemsitry
Staining of tissue sections to see localization and distribution of specific protein
ADV:
-can view localization of proteins under microscope
DIS:
-fixed samples, cannot observe cellular functioning
Plaque assay
measures viral concentration based on infection dose
- used to purify clonal population of virus
- or determine quantity of infectious virus
- cell lysis = viral presence
ADV:
-quantitative: determine viral titres
DIS:
-some viruses don’t cause cell lysis
