In Vitro Experimentation

Question 1

How do you isolate DNA, RNA and proteins - solution based

Answer 1

Starting material - be liquid, cells in suspension, homogenised tissue, blood components e.g. buffy coat

These are lysed, precipitated and separated into supernatant and the protein precipitate

The protein precipitate can undergo treatment to leave RNA, DNA or proteins/lipids

RNA = aqueous phase
Interphase = DNA
Organic phase = proteins, lipids

Question 2

How do you isolate DNA, RNA and proteins - column based

Answer 2

Columns are inserted into a tube and has different affinities

Solution is passed through the column and the target molecule binds to the column

DNA = binds to allprep column, column washed, then DNA eluted

RNA = DNA binds allprep, washed out and into RNeasy column, column washed, RNA eluted

Protein = DNA binds allprep, washed out and into RNeasy column, washed out into new tube and precipitated to form protein

Question 3

How can you measure DNA, RNA and protein quality/quantity

Answer 3

Spectrophotometry

DNA and RNA - 260nm, Protein =280nm
The more light absorbed, the higher the concentration = high OD

Quality
260/280 - pure RNA/DNA = 2.1/1.8, lower ratio = protein contamination

260/230 - pure =1.8, lower ratio - organic contaminants

Absorbance peak = smooth peak shows no contamination

Question 4

How do you isolate DNA, RNA and proteins - column based

Answer 4

Columns are inserted into a tube and has different affinities

Solution is passed through the column and the target molecule binds to the column

DNA = binds to allprep column, column washed, then DNA eluted

RNA = DNA binds allprep, washed out and into RNeasy column, column washed, RNA eluted

Protein = DNA binds allprep, washed out and into RNeasy column, washed out into new tube and precipitated to form protein

Question 5

What methods can be used to analyse DNA

Answer 5

Gel electrophoresis - higher% = smaller fragments = higher resolution

PCR - requires MgCl2, buffer, dNTP, template, primer and polymerase - denature, anneal extension repeat

Sanger sequencing - PCR with fluorescent chain terminators

NGS - DNA extraction, shearing, ligation, library construction, amplification, sequencing, assembly, bioinformatics

Question 6

What is the effect of mutations in noncoding DNA

Answer 6

Regulatory elements (e.g. TATA box) - affects level of gene expression

Spicing of introns - highly conserved GT and AG at the end of introns
Coding sequences lost, or introns kept in mRNA

Question 7

What methods can be used to analyse RNA (differential gene expression analysis)

Answer 7

qRT-PCR - for gene expression analysis, generate cDNA from mRNA followed by PCR, compared to standard curve graph

NMD assay (nonsense mediated mRNA decay) - for mRNA stability

Minigene assay - for splicing variants analysis

Also - Micro-array, and RNA-seq

Question 8

How is qRT-PCR performed

Answer 8

For proteins - reverse transcription to transcribe mRNA into cDNA:
RNA-dependent DNA polymerase assembles cDNA from RNA template
RNase H degrades the RNA template
DNA-dependent DNA polymerase creates the second DNA strand

qPCR:
Uses dNTP labelled so that it fluoresces when bound - output measured real time in thermal cycler
Number of cycles taken to reach threshold value is determined as it is proportional to the amount of starting cDNA (mRNA)

Question 9

What is nonsense-mediated mRNA decay

Answer 9

If mRNA is not found in a sample might be due to NMD

Exon-junction complexes sit at the interaction between exons and intron, marking splice sites
When the ribosome transcribes the mRNA, it will displace the EJC upon encounter
If a premature stop codon is present, the EJC is retained, inducing NMD

NMD occurs via a signalling cascade linked to the EJC which then degrades the RNA

Question 10

How do you prevent NMD/ check if it is occurring

Answer 10

Cycloheximide (eukaryote protein synthesis inhibitor) interferes with NMD through inhibition of protein synthesis and accumulation of mutated mRNA

This is used to view if NMD is occurring indicating a premature stop codon

NMD activity in a particular model can be evaluated by classical RNA quantification methods e.g. qPCR

Question 11

How is the minigene assay performed

Answer 11

They assess the impact of sequence variants on splicing

A genomic segment containing the variant sequence of interest along with flanking intronic sequences is amplified via PCR and cloned into a minigene vector

After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the variant constructs are compared by reverse transcription-PCR analysis and sequencing/gel electrophoresis

This method represents a complementary approach to reverse transcription-PCR analyses of patient RNA for the identification of pathogenic splicing mechanisms

Question 12

What methods are used to analyse proteins

Answer 12

SDS-PAGE - separation of proteins based on molecular weights

Western blot - detection of proteins using specific antibodies

Question 13

How is SDS-PAGE performed

Answer 13

SDS- based solution, Acrylamide gel

Loading dye (density-e.g. glycerol- plus colour-e.g. bromophenol blue), prevents protein floating, and allows visualisation

Visualised by Coomassie blue stain

Proteins negatively charged due to SDS move to a positive electrode
They are separated by size (molecular weight) -smaller proteins move faster

Question 14

How is western blot performed

Answer 14

Blot paper sandwiches a membrane and gel electrophoresis is run

The membrane is then taken to be incubated with the antibody solution

Isolate all proteins from sample → Gel electrophoresis → Transfer to membrane → Block → ELISA; Primary antibody → Secondary antibody

ELISA Immunofluorescence - The primary antibody is designed to bind to the protein of interest, the secondary antibody is designed to bind the IgG

Colorimetric - attached to enzyme, which causes a substrate colour change

Chemiluminescence - attached to enzyme, formation of product releases light, printed on X-ray film

Fluorescence - conjugated with fluorophore which emits light

Question 15

Describe different forms of alternate splicing

Answer 15

Exon skipping or cassette exon - an exon may be spliced out of the primary transcript or retained

Alternative donor site - an alternative 5’ splice junction (donor site) is used

Alternative acceptor site - an alternative 3’ splice junction (acceptor site) is used

Intron retention - a sequence may be spliced out as an intron or simply retained

Mutually exclusive exons - one of two exons is retained in mRNA after splicing but not both

Question 16

What is immunohistochemistry

Answer 16

Cells within a tissue can undergo a similar process as immunofluorescence, however the secondary antibody is conjugated to an enzyme (colorimetric)

This enzyme will react with reagents to create a coloured precipitate where the antibody is bound e.g. horseradish peroxidase

This is seen under a microscope

Question 17

Describe multiplex protein detection - protein assays

Answer 17

Membranes are immobilised with different antibodies and then incubated

There would be two versions - treated and untreated antibody-protein complexes? which are compared
You can then see how a protein is affected by a treatment

Question 18

How do you analyse protein-protein interactions

Answer 18

Immunoprecipitation and Co-IP

This allows you to analyse protein complexes from a mixture by isolating them via an antibody that binds one of the proteins in a complex

An antibody is added to the mixture, followed by a protein A or G coupled bead with an affinity for the antibody

The tube is spun and all the antibodies bound to the beads along with the complex fall to the bottom as an ‘immunoprecipitated’

Question 19

What is Mass Spec and what is it used for

Answer 19

Mass spectrometry is a sensitive technique used to detect, identify and quantitate molecules based on their mass-to-charge (m/z) ratio, in this case proteins

Proteins are extracted from cells/tissues, and digested into peptides

Peptides are attached to support and a laser ionises them and are accelerated through drift tube

Time Of Flight measured– correlates with mass of molecule
Ionization allows scientists to obtain protein mass “fingerprints” that could be matched to proteins and peptides in databases and help identity unknown targets

Question 20

How do you analyse DNA-protein interactions plus give an example of DNA-protein interactions

Answer 20

E.g. Transcription factors

Luciferase reporter assay
EMSA - electrophoretic mobility shift assay
ChIP-Seq

Question 21

Describe EMSA - Electrophoretic Mobility Shift Assay

Answer 21

Determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence
Based on gel electrophoresis

Identifies changes in mobility of fragments

Requires protein, probe, antibody and specific competitor and mutant/non competitor

Lane 1 = 1 probe
Lane 2 = protein + probe - shift is shown as a band is formed somewhere else
Lane 3/4 = protein + competitors - shows if binding is specific
Lane 5 = protein + probe + antibody - super shift proves if it is actually the protein of interest

Question 22

Describe ChIP-Seq to identify TF binding sites (or other protein-DNA interaction)

Answer 22

Chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing

• Steps
	○ Transcription factors bound to DNA in nucleus
	○ Fix the protein to the DNA, then shear
	○ Immunoprecipitate TF of interest, along with attached DNA with Ab complementary to protein Sequence the DNA to determine the binding site

Question 23

Describe the use of luciferase for TF DNA-RNA interactions

Answer 23

TFs are an example of DNA-protein interaction

Luciferase reporter assay for transcription factor activity

Requires a plasmid which expresses the luciferase enzyme when TFs bind its promoter

The reaction of the enzymes releases light
If light is seen, it means TFs bound its promoter to induce transcription

Question 24

What are the cell types used in in vitro techniques

Answer 24

Primary cells
Cell lines
Stem cells

Question 25

What are primary cells and their pros/cons

Answer 25

Primary cells - isolated directly from human/animal tissue, non-transformed, non-immortalised

Limited life-span, retain cell identity, closer to an in vivo model

Pre-characterised and ready to use

Study cells with varied donor characteristics

Some pathways can only be studied in primary cells
Can be used as normal controls for primary diseased cell studies

Question 26

What are cell lines and their pros/cons

Answer 26

Cell lines - derived from tumours or ‘immortalised’ primary cells, passaged over long periods of time

Infinite lifespan, lose cell specificity

Can be immortalised via specific culture conditions, or addition of other genes (oncogenes)

Possess many/some characteristics of the original tissue but may not be entirely representative

From a vial with high mutations and clonal selections - authentication required before use
Study single donor repeatedly

Question 27

What are stem cells and their pros/cons

Answer 27

Stem cells - unlimited self-renewal to produce progeny exactly the same as the originating cell

Embryonic or adult/induced pluripotent stem cells
Can transform into any cell - can act as a repair system to replenish damaged cells

Question 28

What do you need to consider when selecting the appropriator cell line

Answer 28

Species/organ-specific cultures

Functional characteristics - e.g. liver and kidney derived cell line suitable for toxicity testing

Finite or continuous - finite cell lines express the correction functions, while continuous cell lines are easier to clone and maintain

Growth conditions and characteristics - cell culture with a fast growth rate can be chosen for the expression of a recombinant protein in high yields

Question 29

What are the applications of cell cultures

Answer 29

To study;

Normal physiology and biochemistry of cells e.g. metabolic studies

Effects of drugs and toxic compounds of cells - drug screening and development

Mutagenesis and Carcinogenesis
Large-scale manufacturing of biological compounds e.g. vaccines, therapeutic proteins

Question 30

Compare 2D monolayers and 3D cultures

Answer 30

2D monolayers
Limitations - cells lose their phenotype, lack cell-cell and cell-matrix interactions, cannot mimic cellular functions and signalling pathways as in in-vivo conditions

3D cultures
Spheroids are often formed from cancer cell lines or tumour biopsies
More similar to in-vivo conditions, more realistic biochemical and physiological responses
Tumour organoids have shown to predict how well patients respond to cancer drugs to aid in personalised medicine

Question 31

What conditions are needed for cell culture

Answer 31

Maintain aseptic conditions - sterile handling and storage of cell culture, reagents and media

Biosafety cabinet - personal protection from harmful agents inside, product protection to avoid contamination

Growth medium
Provides nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones
Regulates pH/osmotic pressure

Physiochemical environment
pH = 7.4 for most mammalian cell cultures
CO2 = 5-7% for most cell cultures
Temperature = 36-37C

Question 32

Describe the characteristics of fibroblastic-like cells, epithelial-like cells and lymphoblastic-like cells

Answer 32

Fibroblastic-like cells
Elongated shape, bipolar or multipolar
Grow attached to a substrate

Epithelial-like cells
Polygonal in shape, regular dimensions
Growth attach to a substrate in discrete patches

Lymphoblastic-like cells
Spherical in shape
Grown in suspension (n attachment to surface)

Question 33

What is passaging and the two types

Answer 33

The process of plating cells - adherent or suspension

Question 34

What is adherent passaging

Answer 34

Cells that require a solid surface/substrate for attachment, usually derived from tissues of organs where they are immobile and embedded in connective tissue

Trypsin detaches them, and is then neutralised using media with serum
They are split 1/3 into new plates so that they are less packed

Question 35

What is suspension passaging

Answer 35

Cells that grow in suspension and does not require a surface for attachment

Usually culture of cells from blood
Centrifuged and split into 2 using fresh media

Question 36

How do you count cells

Answer 36

Manual counting - cytometer can help with counting cells

Extrapolate the numbers to apply to the whole culture

Automated cell counting - cytometer inserted into machine

Green circles - highlights viable cells (red = non-viable)

Fluorophores for cell counting - can indicate number of viable cells

Question 37

How do you study cell viability/cytotoxicity

Answer 37

Dye exclusion tests - manual counting via cytometer, where cell solution is mixed with trypan blue
Cells that are blue are dead cells, as the damaged membranes allow it through

Biochemical assays - alamarBlue cell viability protocol
AlamarBlue reagent added to cells, incubated and fluorescence/absorbance is read
Incubation results in change of colour via live cell metabolic action

Question 38

How do you study cell apoptosis

Answer 38

TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick end labelling)

Visualises DNA fragmentation by labelling 3’-OH breaks generated in apoptosis

TdT (terminal deoxynucleotide transferase) inserts modified nucleotides (dUTP) conjugated with Br to the 3’ end of the breaks

Depending on the nucleotide modification, you can visualise via colorimetry or via FACS

Biotin + streptabidin-HRP + chromogenic substrate = colorimetry

BrdU + specific Ab + HRP or fluorescent dye = colorimetric or FACS
Fluorescent dye (direct) = FACS

Question 39

How do you analyse genes involved in disease

Answer 39

Studying cells from patients - a cancer patients tumour or healthy tissue is biopsies, and used to establish a primary cell line

Question 40

What are functional effects of mutations on proteins

Answer 40

Loss of function - reduced activity/complete loss of gene product

In heterozygous state, half normal levels of protein product
Gain of function - increased levels of gene expression/development of new functions

Question 41

How can you generate loss or gain of function mutations

Answer 41

Plasmid transfection (overexpression)

Site directed mutagenesis

Inactivation of RNA by siRNA/shRNA
siRNA (small interfering RNA) - chemically synthesised, 20-25 bp long dsRNA
shRNA (short hairpin RNA) - synthesised within the cell, 19-22 bp dsRNA, linked by a short loop

DNA plasmid transfection or lentiviral transfection
Blocking of splicing or translation by anti-sense RNA (morpholinos)
Genome editing techniques e.g. CRISPR

Question 42

What are the different plasmids used for cell transfection

Answer 42

Cloning plasmids - facilitate the cloning of DNA fragments
Bacterial resistance gene, origin and MCS/MRS (multiple cloning/restriction site)

Expression plasmids - used for gene expression
Promoter, transcription terminator sequence, inserted gene

Gene-knock down plasmids - reduce/silence the expression of endogenous gene
ShRNA, promoter for expression of short RNA

Reporter plasmids - study the function of the genetic elements
Promoter, reporter gene (luciferase, GFP)

Viral plasmids - deliver genetic material into target cells

Question 43

What is cell transfections

Answer 43

Cell transfection - introduction of foreign DNA into the nucleus of a cell, usually transient (3-4 days)

Question 44

What are the methods of cell transfection

Answer 44

Lipofection via liposomes (lipofectamine)

Cationic lipid transfection reagent forms complex with DNA = liposome which enters cell

Calcium phosphate precipitation

Electroporation

Microinjection - however this method has to be done one cell at a time

Question 45

What are the steps for insertion of a plasmid

Answer 45

Promoter → 5’ primer site → multiple restriction site → inserted gene → multiple restriction site → 3’ primer site → origin of replication → antibiotic resistance gene → selectable marker

The promoter is usually enables continuous expression, isolated from CMV
Antibiotic resistance and the selectable marker allow selection of successful transfection

Question 46

Compare transient and stable transfection

Answer 46

Transient transfection - expression of foreign DNA for a limited time (24-96 hours) - no DNA integration

Stable transfection - integration of foreign DNA in the genome of the cells

Use selection markers to be able to select only those cells that have integrated the plasmid
Not all cells are able to do be stably transfected

Question 47

What is the structure of mammalian expression vectors

Answer 47

Contains origin, MCS/MRS and promoter

Stable transfection - select using drug, so only integrated plasmids will survive

Epitope tags are added after the MCS/MRS e.g. Myc/HA - when protein is expressed, the tag is expressed alongside it

This allows you to complete a western blot with a complementary antibody to capture it

Question 48

How do you detect expressed proteins via epitope tags

Answer 48

Raise specific antibodies - time consuming, expensive, variable results

Buy specific antibodies - quick, relatively cheap, usually more reliable

Modifies the DNA so that the expressed protein carries a recognisable tag

Question 49

What is a GFP expression construct

Answer 49

Instead of myc tag or HA tag GFP gene can be used to highlight the target gene via fluorescence

Known as a ‘reporter’ gene to detect and localise a target protein, and check transfection efficiency

Fluorescent proteins come in a variety of colours

The advantage of this is that you can view this in live cells

Question 50

What is site directed mutagenesis

Answer 50

Requires plasmid (DNA template) and primers that contain the mutation you want to create - based on PCR

Mutant strand synthesis - perform thermal cycling to

Denature DNA template > anneal mutagenic primers containing desired mutation > extend and incorporate primers

Digestion of template - digests parental methylated/hemi-methylated DNA found in template only

Transformation - molecule transformed into competent cells for nick repair

Question 51

What is shRNA and what is it used for

Answer 51

Used to mimic loss of function diseases

shRNA - synthesised in cell, dsRNA connected by loop, transcribed and exported to cytosol

DICER removes the loop (siRNA production), siRNA loading to RISC, removal of one RNA strand - Targets mRNA with complementary sequence, cleavage of mRNA and further degradation

Leads to stable knock-down cell lines

Question 52

What is siRNA and what is it used for

Answer 52

Used to mimic loss of function diseases

siRNA - chemically synthesised, dsRNA, transfected into cytosol

Integrated to RISC (RNA induced silencing complex), and the strands separate in this complex - Antisense strand hybridises to the complementary mRNA target - Cleavage of targeted mRNA occurs within RISC, with further degradation by other endogenous nucleases

Transient silencing