In Vitro Experimentation Flashcards
How do you isolate DNA, RNA and proteins - solution based
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
How do you isolate DNA, RNA and proteins - column based
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
How can you measure DNA, RNA and protein quality/quantity
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
How do you isolate DNA, RNA and proteins - column based
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
What methods can be used to analyse DNA
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
What is the effect of mutations in noncoding DNA
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
What methods can be used to analyse RNA (differential gene expression analysis)
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
How is qRT-PCR performed
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)
What is nonsense-mediated mRNA decay
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
How do you prevent NMD/ check if it is occurring
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
How is the minigene assay performed
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
What methods are used to analyse proteins
SDS-PAGE - separation of proteins based on molecular weights
Western blot - detection of proteins using specific antibodies
How is SDS-PAGE performed
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
How is western blot performed
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
Describe different forms of alternate splicing
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
What is immunohistochemistry
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
Describe multiplex protein detection - protein assays
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
How do you analyse protein-protein interactions
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’
What is Mass Spec and what is it used for
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
How do you analyse DNA-protein interactions plus give an example of DNA-protein interactions
E.g. Transcription factors
Luciferase reporter assay
EMSA - electrophoretic mobility shift assay
ChIP-Seq
Describe EMSA - Electrophoretic Mobility Shift Assay
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
Describe ChIP-Seq to identify TF binding sites (or other protein-DNA interaction)
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
Describe the use of luciferase for TF DNA-RNA interactions
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
What are the cell types used in in vitro techniques
Primary cells
Cell lines
Stem cells
What are primary cells and their pros/cons
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
What are cell lines and their pros/cons
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
What are stem cells and their pros/cons
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
What do you need to consider when selecting the appropriator cell line
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
What are the applications of cell cultures
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
Compare 2D monolayers and 3D cultures
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
What conditions are needed for cell culture
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
Describe the characteristics of fibroblastic-like cells, epithelial-like cells and lymphoblastic-like cells
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)
What is passaging and the two types
The process of plating cells - adherent or suspension
What is adherent passaging
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
What is suspension passaging
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
How do you count cells
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
How do you study cell viability/cytotoxicity
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
How do you study cell apoptosis
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
How do you analyse genes involved in disease
Studying cells from patients - a cancer patients tumour or healthy tissue is biopsies, and used to establish a primary cell line
What are functional effects of mutations on proteins
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
How can you generate loss or gain of function mutations
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
What are the different plasmids used for cell transfection
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
What is cell transfections
Cell transfection - introduction of foreign DNA into the nucleus of a cell, usually transient (3-4 days)
What are the methods of cell transfection
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
What are the steps for insertion of a plasmid
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
Compare transient and stable transfection
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
What is the structure of mammalian expression vectors
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
How do you detect expressed proteins via epitope tags
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
What is a GFP expression construct
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
What is site directed mutagenesis
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
What is shRNA and what is it used for
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
What is siRNA and what is it used for
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
