techniques

Question 1

Next generation RNAseq

Answer 1

high throughput transcriptome profiling
used to quantitatively assess expression of RNA transcripts in given cell or tissue
isolation of RNA –> RT into cDNA fragments –> fragments sequenced –> transcriptome assembled using bioinformatic tools

Question 2

RT-PCR + controls

Answer 2

measure quantity of target sequence, enable comparative determination of gene expression in cells
RNA isolated –> RT to cDNA –> amplified by PCR (using heat-stable polymerase, primers, nucleotides, thermal and buffering cycle)
cycle thresholds calculated for each gene and compared between experimental and control cells
controls = negative (no template to ensure no contamination), multiple endogenous genes as controls

Question 3

ChIP-seq + controls

Answer 3

used to determine interactions between proteins and DNA
DNA sample obtained with protein bound
protein cross-linked to DNA
DNA sheared (endonuclease or sanitation) to generate small DNA fragments
immunoprecipitation with antibody specific to protein
cross-linking reversed to release DNA fragments
genome sequencing used to determine DNA sequence of bound fragments = mapped back to original location in DNA
controls = compare ChIP seq peal to same region in matched control, also do mock IP = DNA obtained from IP without any antibody present, or with antibody against protein not known to be involved in DNA binding

Question 4

CRISPR/Cas9

Answer 4

CRISPR/Cas9 is an RNA/protein-mediated cleavage mechanism where a guide RNA hybridizes to the DNA target and brings the double strand endonuclease Cas9 to create a double strand break. Need protospacer adjacent motif (PAM) site: NGG which limits choice of target [2] (Mol med, 2017)

Question 5

TALENs

Answer 5

TALENs are proteins engineered to bind to a specific approximately 15-17 nucleotide sequence and bring a nuclease activity to cleave the DNA (two amino acids recognize each base pair for the sequence specificity). Two TALENs are required to cause the required double strand break [2] (Mol med, 2017)

Question 6

CRISPR vs TALENs

Answer 6

CRISPR uses gRNA, TALENs use proteins
CRISPR is more efficient except in areas of compact heterochromatin
CRISPR easier to design
TALEN = more specific, less risk of off-target effects
CRISPR needs PAM site, TALENs does not = increases target choice

Question 7

surveyor nuclease analysis

Answer 7

used to detect mutations = based on new mismatch-specific DNA endonuclease

1. PCR to amplify target DNA from both mutant and WT DNA
2. hybridisation to form heteroduplexes between mutant and WT DNA
3. treatment of annealed DNA with surveyor nuclease (cleaves with high specificity at 3’ side of any mismatch site in both DNA strands) –> analysis of digested DNA productions using WB or HPLC

Question 8

cytometric bead assays

Answer 8

used to quantify levels of cytokines within the serum
incubated serum with fluorescent beads coated with antibodies against specific cytokine
beads of different specificities have distinct baseline levels of fluorescence that can be distinguished by FACS analyser
cytokines bound to beads are detected by incubation with cytokine-specific secondary antibody labelled with different fluorescent protein
analysed by FACS
shift in fluorescence of beads can be quantified by relating it to the shifts induced using titrated standards of cytokines
provides info on absolute levels of cytokines but not cellular source

Question 9

ELISpot

Answer 9

quantitatively measures cytokine secretion from single/known number of cell(s)
coat well with antibodies specific to cytokine
block to prevent NSB
incubate cells with/without stimulus to activate cytokine secretion
cytokines will attach to antibody adhered to wall
wash to remove cells and other substances (after 24 hours)
add cytokine-specific biotinylated secondary antibodies = bind to cytokine attached to primary antibodies
add substrate = forms insoluble precipitate that forms spots of chromogen deposited in wells
spots read by automated ELISPpot reader or counted under microscope
number of spots - precursor frequency, diameter = proportional to quantity of cytokine produced

Question 10

use of tetramers

Answer 10

used to detect and enumerate antigen-specific T cells
composed of 4 MHC molecules bound to peptide epitope derived from antigen of interest
biotinylation of molecules permits assembly into tetrameric complex when mixed with streptavidin
conjugation to fluorescent marker allows for detection by FC
affinity of single peptide-MHC interaction for TCR is too low to permit stable binding, avidity of tetramers is sufficient to label cells expressing appropriate TCR

Question 11

GFP KI mouse

Answer 11

GFP reporter gene expressed under promoter of desired gene

allows cells up regulating gene of interest to be easily detected by flow cytometry = produce green fluorescence

Question 12

intracellular cytokine staining

Answer 12

stimulate T cells in vitro using peptide pools and brefeldin A (prevents secretion of cytokines synthesised de novo so they accumulate in intracellular compartments)
permealise cells to permit entry of cytokine-specific antibodies
stain with antibodies conjugated to fluorophore
limitations = no temporal information (snapshot of TC activity immediately after stimulation, at best semi-quantitative (cannot relate MFI to known quantity of cytokine)

Question 13

using CD45.1 and CD45.2

Answer 13

can transfer T cells expressing 1 isoform of CD45 (e.g. CD45.2) into animal expressing alternative isoform (CD45.1)
can then identify transferred cells using antibody specific to CD45.2 that does not cross-react with CD45.1

Question 14

CSFE/Celltrace violet + assumptions

Answer 14

cell division dye used to quantify proliferation
labels cytoplasmic proteins
as cells divide, cell trace violet is divided between daughter cells
fluorescence intensity halves with each division, progressively diluted out
can quantify fluorescence to measure how many times cells divide
assumes that: labelling is not toxic and doesn’t affect physiology/function of cells
assumes fluorescence intensity is stable in vivo over observation period and not progressively degraded over time

Question 15

IHC

Answer 15

Immunohistochemistry/immunofluorescence
Need to select several fields of view
Selection should be blinded  ensure no bias from
the researcher unintentionally/intentionally altering the data
Need to quantify fluorescence
Is staining heterogenous?
Have they described what the different colours represent in the methods?
Have they confirmed results with another technique? = e.g. western blotting, flow cytometry
Have they said where section is taken from?
Limitations = specificity of antibodies, false positives, possibility of artefacts caused by epitope-tagged proteins

Question 16

IC50

Answer 16

IC50

• Half-maximal inhibitory concentration = measures potency of substance in inhibiting biochemical function

Question 17

reducing vs nonreducing

Answer 17

• Reducing agents disrupt disulphide bonds

* Need to make sure to use non-reducing agents if looking at dimeric/trimeric structures

Question 18

isotype control

Answer 18

• Primary antibodies that lack specificity to target but match class and type of primary antibody used in application
• Used as negative controls to help differentiate between non-specific background signals and specific antibody signals

Question 19

cre recombinase

Answer 19

Enzyme that catalyzes site-specific recombination of DNA between loxP sites

Question 20

PFU

Answer 20

Measure of the number of virus particles capable of forming plaques per unit
volume

Question 21

FACS

Answer 21

Single cells, isolated in individual droplets are analysed in succession at high speed, are illuminated with lasers and light detected; one or more specific fluorescently labelled reagents (usually mAbs), together with scattering of light, enable detection and quantification of populations of cells; cells can be sorted.
Sorting works by imparting different types of charge on the individual droplets (based on the properties detected on the cell contained in the droplet), so that the particles can be deflected into a different collection tubes.

Question 22

flow cytometry

Answer 22

???

Question 23

xenograft vs synergeneic transfer

Answer 23

xenograft = transfer between different species
Human tumour xenograft = transplant human tumour cells (under the skin or into organ of tumour origin) into immunocompromised rodents that do not reject human cells

synergeneic = transplant tumour cells from genetically identical/sufficiently identical + immunologically compatible into recipient so as to allow transplantation without rejection
graft is called isograft

Question 24

mass spec

Answer 24

mass/charge spectrum produced by individual peptides are compared to reference spectrum to identify peptide
searching human database of peptide derived from human proteins, an algorithm can identify candidate proteins in original sample

Question 25

plaque

Answer 25

A plaque is a small (very roughly 1 mm diameter), circular area of cytolysis in a monolayer of susceptible cultured target cells in which a single infectious virion initiated an infectious cycle. From a dilution series one can measure the titre of infectious virions in a sample.

Question 26

BrdU staining

Answer 26

BrdU = thymidine analogue, incorporated by cells undergoing DNA replication incubate cells with BrdU, then add anti-BrdU antibodies and fluorescently labelled secondary antibodies w 2nd stain for DNA (e. g. 7AAD, PI) low BrdU, small amount of DNA = dead (DNA compacts during apoptosis) low BrdU, normal DNA content = G0/G1 high BrdU, increasing DNA = S phase high BrdU, double DNA = G2/M phase can be toxic and interfere with biology, cells must be permealised and DNA denatured

Question 27

annexin V staining

Answer 27

annexin binds PS, presented in outer leaflet of CSM following apoptosis dead cells have membranes permeable to annexin so produce false positives label with fluorophore also add 2nd impermeant dye (e.g. PI) = only permeates dead cells annexin and PI = dead annexin, no PI = apoptotic neither = live

Question 28

exome sequencing = sequence enrichment strategies

Answer 28

array-based = oligonucleotides capture regions of interest and fix on surface, genome DNA sheared, fragments undergo end-repair and amplified using PCR In solution = oligonucleotide probes added to fragmented DNA sample and selectively hybridise with regions of interest, beads pulled down

Question 29

luciferase reporter

Answer 29

A target gene’s promoter region (G13 in this case) is encoded on a plasmid which is linked to a reporter in the form of firefly luciferase. When the promoter region is bound by a transcription factor the reporter gene, luciferase, is transcribed and expressed. This leads to emission of light when lysates of cells expressing the plasmid are mixed with a fluorogenic substrate. The amount of light is directly proportional to the amount of target gene transcription, and is thus a quantitative assay of transcription factor binding.

Question 30

western blot

Answer 30

Need homogenized sample to make protein available for antibody to bind. Use gel system to separate proteins according to charge and molecular weight. After transferring to another membrane the antibody is added, and detected as a bands using another label (e.g. horseradish peroxidase or fluorescent secondary Ab); must say protein, antibody and molecular weight [2 marks] (CRR, 2017)

Question 31

proteomics

Answer 31

proteins analysed by mass spectrometry proteins digested into fragments and mass determined abundance is estimated by determining mean intensity of peptides from protein

Question 32

TUNEL staining

Answer 32

used to detect apoptotic cells, that have undergone extensive DNA degradation during later stages of apoptosis uses TdT enzyme that can attach nucleotides to blunt ends of DSB use nucleotides conjugated to fluorophore, biotinylated so detected by HRP streptavidin and DAB, or bound by fluorescently/chemically labelled mAbs imaged by light microscopy or flow cytometry sensitive and fast later stages detected than annexin, false positives as DNA degradation can be caused by things other than apoptosis, need to fix and permealise samples

Question 33

TaqMan

Answer 33

used to quantify RT-PCR use oligonucleotide probe labelled with reporter fluorophore = emits fluorescence at a wavelength absorbed by quencher fluorophore DNA polymerase displaces and degrades probe during elongation reporter released from quencher = fluorescence emitted

Question 34

SYBR green

Answer 34

fluorescent dye only activated on intercalation into DNA sequence (used in RT-qPCR)