Prof. Benfante Flashcards
How cell culture is used for?
- Total RNA Extraction -> RT-PCR
- Transfection -> Reporter Genes -> Promoter analysis
What are the sources of contamination when working with RNA?
- Endogenous => cell lysis
* Exogenous: hands, durst
What is DEPC?
DEPC: Diethyl dicarbonate
DEPC can absorb all the RNA in water
Why we use DEPC-treated water?
DEPC-treated water inactivates DEPC, therefore we obtain RNase free water
How can we create RNase-free environment?
1) Treatment with DEPC when possible -> it’s not possible for TRIS containing solutions
* TRIS: Tris base is used in buffers like TAE buffer. It increases cell membrane permeability
2) Inhibitors:
- isolated from human placenta
- they form an enzymatically inactive complex
- commercial
3) Denaturing agents in lysis solution:
* guanidine-HCl:
- strong chaotrope
- strongest denaturant
- used in protein folding
- decreases enzyme activity
- increases solubility of hydrophobic molecules
- guanidium thiocyanate + beta-mercaptoethanol:
- reducing agent -> irreversibly denatures RNases by reducing disulphide bonds
What are the studies that use RNA?
- Synthesis
- Maturation
- splicing
- 5’end
- 5’-3’ exonucleolytic degradation
- 3’end
- Stability
- polyadenylation
- Translation
What are the techniques RNA can be used in?
- Northern Blot
- S1 mapping
- RNase-protection
- Primer extension
- RT-PCR
- Real-Time PCR -> most used
(they measure steady-state level)
- Run-on -> it measure changing in steady-state due to transcription regulation
What is be obtained from RNA extraction?
- Total RNA
- Cytoplasmic RNA
- Nuclear RNA
How RNA extraction is performed?
1) Lysis in the presence of non-ionic detergents (NP-40) -> Proteinase K -> DNase-1
2) Organic Solvents (acid phenol)
3) Precipitation to separate RNA from other nucleic acids
4) Centrifugation by density gradients (CsCl) -> RNA pellet
5) Column purification (Kit)
What are the steps of Guanidinium Thiocyanate RNA Extraction?
1) Homogenization/Lysis: by Guanidium phenol -> RNase is not working
2) Phase Separation: by Chloroform
* Separated phases are: Aqueous phase= RNA, Interphase=DNA, Organic phase=Protein (from top to bottom)
3) Extraction/Precipitation: by Isopropanol
* you get RNA pellet
4) Resuspension: by Water/TE
What are the two types of Phenol Extraction?
Traditional Phenol Extraction can be acidic or basic.
- Basic:
both RNA and DNA are in the aqueous phase, protein is in organic phase - Acidic:
RNA will remain in aqueous phase but DNA is in interphase, protein is in organic phase.
Therefore, you can separate them in one step - this is what we used
What are the differences between DNA extraction and RNA extraction?
- DNA Extraction:
- pH=8
- Reagents are NOT prepared with DEPC-treated water
- DNA can be extracted prior and is stored in batches
- Long-term storage is at -20°C
- Steps:
i) cell lysis or breaking the cell membranes
ii) removing the membrane lipids
iii) precipitating DNA - RNA Extraction:
- pH=4.7
- All reagents are prepared with DEPC-treated water
- RNA extraction is done just before the downstream procedures
- Long-term storage is at -80°C
- Steps:
i) cell lysis
ii) guanidium thiocyanate-phenol-chloroform extraction
iii) preparation with isopropanol
Why column kit is used in RNA extraction and purification?
Column kit is very pure and fast. It is ready for PCR, especially for quantitative PCR
What are the steps of RNA extraction and purification with standard RNA kits or column kits?
1) Lysis
2) Homogenization by filtration
3) RNA binding
4) On-colun rDNase digest
5) Washing
6) Elution
Why we are using Quantitative PCR: RT-PCR ?
- To see if our gene of interest is present
- To se if there is a change in the level of our gene due to ean experimental condition
- To see in what degree our treatment changes the expression level of our gene of interest
What are the steps of Quantitative PCR: RT-PCR ?
RT-PCR has 2 steps:
Step-1: Reverse Transcription= Retro Transcription
Step-2: PCR, amplification
What are the types of Reverse Transcriptase-PCR?
Reverse Transcriptase-PCR can be performed with 3 different types of primers:
- Random primer
- Oligo (dT) primer
- Sequence-specific primer
Why you need to denature RNA?
Because RNA is single-stranded and it has multiple secondary structures
What are the steps of PCR?
1) Denaturation
2) Annealing
3) Extension
What are the important parameters for PCR?
1) DNA to amplify:
because of GC content => GC content should be 40-60% with the 3’ending in G or C to promote binding = GC Clamp. Because G and C bases have stronger hydrogen bonding and help with the primer stability
2) Primers: because of their length
3) TAQ DNA Polymerase:
because it has 5’-3’ proofreading activity but no 3’-5’ exonuclease activity
4) dNTPs
5) Enzyme concentration
6) Mg2+:
most important parameter because it is cofactor of DNA Polymerase => boosting DNA amplification
*if it’s too high => non-specific bindings are increased => errors in DNA replication
7) Thermocycler
What are the Applications of PCR?
- Basic Research
- Cloning, sequencing and modification of gene sequences
- Evolution studies
- Medicine
- Pre and post natal diagnosis of genetic diseases
- Tumour diagnosis
- Infectious disease diagnosis
- Forensic Medicine
- Paternity
- Suspect individuals identification
- Industry
- Identification of Genetically Modified Organisms (GMO)
What are the PCR Extensions?
- Long Accurate PCR
- Nested PCR
- Reverse Transcriptase PCR
- Real Time PCR
What are the some applications of Quantitative PCR?
- Quantitation of gene expression
- Copy Number Variation (CNV)
- Genotyping
- Single Nucleotide Polymorphism (SNP) genotyping
- GMO (Genetically Modified Organisms) detection
- Drug target validation
What are the differences and advantages/disadvantages of qPCR (Quantitative PCR) chemistries?
- TaqMan probe
- easy to design as exon-exon junction => it’s NOT genomic
- avoids signals from contamination with genomic DNA
- direct proportion between fluorescent signal and DNA amplification
- it is 15 bp and it has 5’fluorophore (reporter) and 3’quencher
Advantages:
- increased specificity
- use when the most accurate quantitation of PCR product accumulation is desired
- option of detecting multiple genes in the same well = multiplexing
Disadvantages:
- relative high cost of labeled probe
- SYBR Green
- it intercalates with minor groove of the DNA, meaning that it only intercalates with double stranded form
Advantages:
- Relative low cost of primers
- no fluoorescent-labeled probes required
Disadvantages:
- less specific
- not possible to multiplex multiple gene targets
What are the parameters for designing Taqman probes (primers)?
1) Tm (melting temperature):
Primer Tm= 58-60°C
Taqman Tm= +10°C than primer Tm
2) Length:
15-30 bp
3) GC content:
30-80%
How to measure fluorescence?
Relative Quantification: “Delta Delta Ct (∆∆Ct)” Method
1) Normalize ∆Ct of the target gene to the reference gene (exogenous control, housekeeping gene) is calculated for each sample:
∆Ct= (Ct_target) - (Ct_reference)
*logarithmic => substraction
2) Normalize the ∆Ct of the test sample to the ∆Ct of the calibrator
∆∆Ct= ([(Ct_target) - (Ct_reference)]_test) - ([(Ct_target) - (Ct_reference)]_calibrator)
*second part (calibrator) is equal to 1
3) Calculate the fold difference in expression
2^(-∆∆Ct)= normalized expression ratio
What is SNP genotyping is used for?
Allelic discrimination assay
- it is performed by using 2 Taqman probes: VIC and FAM
Steps:
1) Assay components and DNA Template
2) Denatured Template and Annealing Assay Components
3) Polymerication and Signal Generation
What is Cell Culture?
It refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.
The cells may be removed directly from the tissue and disaggregated by enzymatic or mechanical means before cultivation, or from biological fluids, such as blood.
They may be derived from a cell line or cell strain that has already been established.
Cells are grown in the presence of factors and metabolites useful to their growth.
What are the applications of tissue culture?
Tissue culture application can be divided into 2 categories as basic and applied.
- Basic:
- Intracellular activity
- Intracellular flux
- Genomics
- Proteomics
- Cell-cell interaction
- Applied:
- Cell products
- Immunology
- Pharmacology
- Tissue engineering
- Toxicology
What are the advantages and disadvantages of growing animal cells in culture?
- Advantages:
- allows specific cell types of be studies free of the influence of surrounding tissues in the intact animal
- provides more control over experimental conditions
- can mimic cell-cell and cell-ECM (extracellular matrix) interactions seen in tissues
- clonal colonies can be generated in 2 weeks
- defined, serum-free medium formulations are available for some cell types
- Disadvantages:
- question of cell behaviour in culture vs. in tissues
- can be difficult to grow or to maintain consistent growth conditions from one experiment to another
- growth medium is more complex
- required essential amino acids, vitamins, serum (hormones, growth factors etc.)
What are the animal in vitro cell culture classes?
Animal in vitro cell cultures divided into 2 classes: primary cultures and continous cell lines
Primary Culture:
* Advantages: best representation of cell behaviour in normal tissues
- Disadvantages:
- have a finite life span
- undergo replicative senescence after 50-60 doublings
- required several preparation for long-term project
- Cell types commonly prepared:
- fibroblast (skin)
- myoblast (skeletal muscle)
- leukocytes (blood)
- lymphocyte (blood)
Continous Cell Line:
- they have acquired one or more genetic mutations that allow them to escape senescence => cells become immortal (transformation)
- it can occur spontaneously or can be chemically or virally induced
- when a finite cell line undergoes transformation and acquired the ability to divide indefinetely => it becomes continuous cell line
- Advantages:
- can grow indefinetely in culture
- more reproducible results
- Disadvantages: often they are less phenotypically related to the source tissue
What are the culture conditions?
They vary for each cell type, but the artificial environment is invariably consisted of:
- medium: supplies the essential nutrients such as amino acids, carbohydrates, vitamins, minerals
- growth factors
- hormones
- gases: O2, CO2
- regulated physico-chemical environment: such as pH, osmotic pressure, temperature
- pH: 7.2-7.4
- buffer (NaHCO3): to maintain pH constant
- temperature: 35-37°C
- CO2: 5-10% -> it influences the pH
How can you control the pH?
A pH sensitive dye that allows to check pH during cell growth can be added to the medium.
One example of pH sensitive dye is Phenol Red and it has different colors for different pH levels
- Phenol Red
- orange/red => pH=7.3
- red/purple => pH: alcaline
- yellow/orange => pH=acidic
What are the Culture Mediums?
Commercially available media contain all the growth factors
The most common basal media:
- MEM: Minimum Essential Medium
- DMEM: Dulbecco’s Modification of MEM
- RPMI: Roswell Park Memorial Institute
- They differ for aminoacids, salts and glucose concentration.
- Basal medium is stored at 4°C
- Basal medium is complemented with some factors:
- Glutamine: essential aminoacid, very labile
- Antibiotics: penicillin/streptomicin
- Serum: it contains all the growth factors
What does Culture Medium contain?
- Inorganic Salts -> Buffer
- essential for cell growth and for the maintenance of cellular functions
- they have a buffer function to prevent pH variation due to changing environment conditions and/or catabolism products
- Vitamins
- they act as catalyzers or as substrates to facilitate or control some metabolic functions
- Proteins
- necessary for protein synthesis
- Carbohydrates
- they represent the main source of energy or carbon for biosynthetic pathways
What is Serum and what is it used for?
Serum is a complex mixture of proteins and fundamental elements for in vitro growth of most of the cells
It contains growth factors, adhesion factors and other elements.
- Growth factors: PDGF, EGF, IGF
- Adhesion factors: fibronectin, vitronectin
- Other elements: transferrin, albumin, cholesterol, fatty acids and glucocorticoids, minerals
Fetal Bovine Serum (FBS) is one of the most used serums.
What are the different types of growth?
Growth can be in adhesion or in suspension according to cell type.
Adhesion:
it requires the interaction between membrane receptors with adhesion proteins on plate surface.
* plates, multiwells and flasks can be used
- neuron cells
- epithelial cells
- mesenchymal-derived cells
* in vivo belonging to solid tissues
Suspension:
cells grow floating in the culture medium.
- hematopoietic-derived cells
* usually present in biological fluids
What are the Cell Morphology Categories?
- Fibroblast-like:
- cells are bipolar or multipolar
- cells have elongated shapes
- cells grow attached to substrate
- Epithelial-like:
- cells are polyonal in shape with more regular dimensions
- cells grow attached to a substrate in discrete patches
- Lymphoblast-like:
- cells are spherical in shape
- cells usually grown in suspension without attaching to a surface
What are the growth supports for adhesion growth?
usually polystirene:
- rigid material
- good resistance to aqueous solutions
- limited resistance to solvents
- not toxic for cells in culture
- the surface is trated to create an hydrophilic environment (negative net charge) to favour stable bound with adhesion factors in the serum
- covered with matrigel, polylisine, fibronectin, gelatin
What is Plating Efficiency?
A plating efficiency is a measure of the number of colonies originating from single cells. It is a very sensitive test and is often used for determining the nutritional requirements of cells, testing serum lots, measuring the effects of growth factors, and toxicity testing.
Cells generally need a minimum density to grow efficiently
- General rule:
density > 10^4 cell/cm^2 - it should not be too much or too little
What is Duplication Rate?
The time it takes for a population to double its size.
- cells grow exponentially
- the doubling time varies among cell lines:
- about 20-24 hours for animal-derived cell lines
- about 24-30 hours for human-derived cell lines
What is subculturing?
Subculturing, also referred to as passaging cells, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.
Subculture is used to prolong the life and/or expand the number of cells or microorganisms in the culture
How to calculate the cells when subculturing?
For example:
If you are resuspending the cells in 10 ml => 10ml/3/10= 0,33ml
You have cells in 75cm^2 area with 100% concentration, and you want to passage your cells to a 25cm^2 area with 10% concentration.
Then, your dilution factor for area is 3, and your dilution factor for concentration is 10.
Since, final area is 1/3 of the initial area, (1/3)^2=1/9 of the cells should be passed.
You want to pass your cells 1/5 ratio
You have to 1/9 of cells
What do you need differently from suspension cell culture when subculturing adherent (monolayer) cells?
Adherent cells, grow attached to a surface such as the bottom of the culture flask. These cell types have to be detached from the surface before they can be subcultured.
So, you need a dissociation reagent when passaging adherent cells such as trypsin. Suspension cell culture does not require enzymatic or mechanical dissociation.
You use Trypsin/EDTA Solution because chelation of Calcium and Magnesium ions is needed for adhesion.
Trypsin is an proteolytic enzymed which is derived by pig pancreas.
It degrades the extracellular matric (ECM) proteins that maintain cells adherent to the substrate.
How do you count cells? What are the methods?
There are several methods for counting cells.
One very simple and saving method is the Hemacytometer or known as Burker Chamber.
Burker chamber is divided into 9 squared fields, in turn subdivided into 16 squared fields.
Tryphan blue dye is used for the counting because it allows us to distinguish dead and living cells. Dead cells appear blue, living cells appear clear because the dye passes through the membrane of dead cells.
Counting process:
1) choose some squares from 9 big squares
2) count the viable (living) and non-viable (dead) cells in those squares
* you need to set a rule about the counting the cells that are in the edges of the squares. For example, you only count the cells that are in the top and left edges.
Calculating the number of cells:
1) Percentage of viable cells:
100x(number of viable cells)/(total number of cells)
2) Average number of cells per square:
(viable cells)/(number of squares you counted)
3) Dilution factor:
(final volume)/(volume of cells)
4) Concentration (viable cells/mL):
(average number of cells/square)x(dilution factor)x(10^4)
What is Gene Transfection?
Transfer of exogenous DNA in mammalian cells to study the function of the genes and the regulation of their expression.
What are the applications of transfection?
- in Research:
a) cell culture:
i) identification of regulatory elements
ii) gene structure and function
iii) protein factory: - study
- drugs
b) in vivo:
transgenic animals generation - models to study the molecular basis of diseases
*in Clinical Practice:
Gene Therapy
- restoring metabolic functions
- increasing natural defence
What are the types of Transfection?
There are 2 types of transfection: Transient and Stable.
1) Transient Transfection:
- short-term experiments (ICC=immunocytochemistry, promoter analysis)
- cells are harvested 48-72 hours after transfection
- gene over-expression
- not uniform cell population: few cells with a lot of plasmid
- immediate answer
2) Stable Transfection:
- long-term experiments
- possibility to isolate and expand single clones expressing the transfected DNA
- plasmid DNA integrates in the genome randomly
- a selection marker is required (resistance to antibiotics…)
- uniform cell population: many cells with little plasmid DNA
- always express the gene of interest -> plasmid DNA with marker that will select only the ones who takes up plasmid
=> you will get clone
=> multiple apperance
=> more than one clone
What are the characteristics of Transfection Methods?
- highly efficient
- low toxicity
- reproducible in vitro and in vivo
What are the processes of transfection methods?
- introduction of DNA into the cell
- gene must be expressed
- selection of stably transfected cells
- characterization of the produces protein
What is the problem with transfection methods?
Problems with figuring out how to overcome natural barriers
Natural barriers:
- DNA: very polar => negative charge
- lipophilic cell membrane
What are the types of Transfection Methods?
There are 2 types of transfection methods: Chemical and Physical.
What is the Chemical Transfection Method?
Liposomes
- Mixture of polycationic and neutral lipids, it allows the formation of unilamellar liposome vesicle with a positive net charge
- The cationic group associates to the negative P-group of nucleic acids
- DNA-lipid complexes fuse with cell membrane and the content is released inside the cell
- Advantages:
- low toxicity
- Disadvantages:
- difficulty in producing liposomes
- high variability
What is an example for Chemical Transfection Method?
FuGENE-HD:
FuGENE® HD is a 100% synthetic, multi-component, non-liposomal transfection reagent designed for the delivery of DNA into more challenging eukaryotic and insect cell lines.
FuGENE® HD interacts with nucleic acids & the cell’s membrane to provide efficient and safe entry into the cell.
This mechanism allows users to overcome barriers in difficult-to-transfect lines such as primary cells, stem cells, and suspension cells.
- Advantages:
- simple
- efficient
- low toxicity
- Disadvantages:
- affected by the presence of antibiotics and/or serum
What are the Physical Transfection Methods?
- Electroporation
- Microinjection
- Gene Gun
What is Electroporation?
What are the advantages and disadvantages of it?
Electroporation:
Cells are mixed with DNA and subjected to a short electrical pulse.
This allows the formation of pores in the cell membrane
=> electrical pulse -> push DNA
Advantages:
- useful with cells that are not transfected by chemical methods
- DNA enters directly without passing by the endosomal compartment where it can be degraded
Disadvantages:
- intensity and duration of electric pulse must be optimized
- highly toxic => 50% of cells die
What is Microinjection?
What are the advantages and disadvantages of it?
Microinjection:
DNA is introduced into the cell (in the cytoplasm or in the nucleus) by a glass capillary, under high pressure.
Due to the reduced size of the cells, microinjection required a high sophisticated apparatus that include:
- a microscope
- a micromanipulator
- an injector
Advantages:
- transfer DNA directly into the cytoplasm or into the nucleus
- expression efficiency is increased
- expression level can be easily modulated
Disadvantages:
- technically difficult
- few cells at a time
- expensive
What is Gene Gun?
What are the advantages and disadvantages of it?
Gene Gun:
Gold particles cover with DNA are shot under Helium high pressure directly onto the cells or tissues.
* usually used in plant or muscles
Advantages:
- in situ transfection
- feasible for cell system that is hard to transfect with other methods, especially in vivo
- gene expression studies under normal physiological conditions are permitted:
- tissue specific expression
- development
- less DNA and less cells can be used
Disadvantages:
- invasive
- efficiency strictly dependent on cell type
- many parameters to control
- expensive
What are the Optimizations of the process?
- DNA
- no contaminants: proteins or RNA
- sterile solutions
*Cell type
- Plasmid vector
- mammalian expression vector
- Transfection time
- from 30 minutes to 4 hours
- Cell culture medium
- serum and antibiotics effects
What is a vector?
Vectors can be plasmids or virus (infection).
Plasmids are circular and they have promoter, Multiple Cloning Site (MCS), cDNA, replication signals (cis or trans), selective markers, and ORI (origin of replication. usually SV40)
Promoter:
i) short nucleotidic sequences
ii) start of transcription
iii) Enhancer: positive regulatory elements
*derived from virus
- SV40 early gene enhancer: function in many cell types
- LTR: Long Terminal Repeat
> Raus Sarcoma Virus
> CMV
iv) Silencer: negative regulatory elements
What are the differences between Selectable Marker and Reporter Gene?
Selectable Marker:
- a gene whose expression allows one to identify cells that have been transformed or transfected with a vector containing the marker gene
- helps to distinguish between transformants and non-transformants
- has its own promoter
- can be in the plasmid or within the gene construct
- transformed cells with selectable marker can grow in the selectable medium but, the non-transformant cannot grow since they lack the selectable marker
- Examples: antibiotic resistance genes, antimetabolic marker genes, and herbicide resistance genes
Reporter Gene:
- a gene used to “tag” another gene or DNA sequence of interest, such as promoter
- helps to measure the amount of expression of the transformed gene
- regulated under the promoter of the transformed gene
- in between the promoter and the gene of interest
- amount of gene product in the reporter gene depends on the amount of successfully-transformed genes of interest
- Examples: GFP and Luciferase
What are the types of Reporter Genes?
- Green Fluorescent Protein (GFP)
- Red Fluorescent Protein (RFP)
- Yellow Fluorescent Protein (YFP)
- β-Galactosidase
- β-Lactamase
- Luciferase -> not found in mammals
- Chloramphenicol Acetyl Transferase
What are the applications of reporter genes?
- in vitro drug screening
- intracellular drug screening
- high throughput screening
- in vivo parasite monitoring
- whole animal/organ imaging
- in vivo drug screening
- vaccine efficacy testing
- gene expression studies
- protein colocalization studies
What are the features of Green Fluorescent Protein (GFP)?
- expressed in jellyfish Aequorea Victoria
- it produces bioluminescence due to energy transfer (calcium dependent process associated to Auquorine protein)
- exposure to UV light generates green autofluorescence in the absence of calcium, aequorine or any cofactor or substrate
- point mutations in the chromophore part of the protein give rise to GFP variants that emit light at different wave length
- CFP (cyan)
- YFP (yellow)
- BFP (blue)
The GFP from has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm.
Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum.
What are the features of Chloramphenicol acetyl-transferase?
- Bacterial enzyme: resistance to chloramphenicol antibiotic
- acetylation of chloramphenicol
- C14 chloramphenicol used as substrate is acetylated only in the transfected cells where the promoter controlling its expression is active. The radioactive molecules can be revealed by TLC or autoradiography
What are the features of ß-galactosidase?
- codified by LacZ gene of E.Coli
- conversion of substrate in colored compounds
- spectroscopy: degradation of ONPG (o-nitrophenyl-b-Dgalactopyranoside) -> yellow soluble compound
What are the features of Luciferase?
- proteins expressed in different organisms but not in mammals
*Firefly Luciferase (FLuc):
Luciferin + ATP + O2 -> Oxyluciferin + AMP + light
*Renilla Luciferase (RLuc):
Coelenterazine + Na + O2 -> Coelenteramide + light + CO2
What are the different applications of reporters?
Reporters can be used for a variety of applications:
a) Gene Expression:
- Transcription and post-transcriptional regulation
- promoters/response elements
- enhancers
- 5’ and 3’-UTRs
- transcription factors
- RNA binding proteins & miRNAs
- Post-translational regulations
- protein stability
- protein localization
- protein-protein interactions
b) Cellular Events:
- Receptor activation/signalling
- receptor ligands, agonists & antagonists
- nuclear receptors
- Pathway analysis
- defining pathways
- protein-protein interactions
- Disease/immune responses
- cellular response to infection
- cellular response to therapy
- infectious agent replication/response to therapy
What are the Promoter types?
- Constitutive
- Inducible
- Tissue-specific
*promoters only works in humans
What are the ideal features of the reporter genes?
- They do not have a similar function of the gene of interest
- The reporter activity must not interfere with physiological processes: the signalling pathways and cell metabolism must not be affected
- Detection with high sensitivity
- Low endogenous background
- There should be quantitative assay
- Reporter activity assay must be specific, reproducible and not time-consuming
What are the applications of Luciferase Reporter Assays?
- promoter studies
- gene regulation
- cell signalling pathways
- compound screening
- bioassays
- protein interactions
- post-translational modifications
- virus-cell interactions
How to design a reporter assay?
1) Cloning of reporter vectors
2) Transfection & cultivation
3) Signal development & detection
How to study a promoter?
a) Deletion construct:
* by measuring it, you can find the important part
1) mapping transcription start site (TSS)
2) making deletion contructs
3) cloning and/or subcloning
4) transfections
5) Luciferase assay
6) data plotting
7) data interpretation
b) Dual assay format:
* measurement of 2 luciferase reporters in a single sample
1) transfection
2) cultivation 2-3 days
3) treatment
4) Dual-Luciferase Assay
I) addition of assay reagent for luciferase reporter-1
II) measurement of luciferase reporter-1
III) addition of Stop&Glo reagent
IV) measurement of luciferase reporter-2
What is the aim of the lab experiment that is performed?
The aim of this experiment is to observe the expression of GAPDH gene in HeLa cells.
It is done by extracting the RNA of the cells and performing retrotranscription, followed by PCR and analyzing with gel electrophoresis.
What are the steps of the experiment?
Step-I) Total RNA Extraction
1) Homogenization/Lysis
2) Phase separation
3) RNA Precipitation
4) RNA Wash
5) RNA Solubilization
6) RNA Quantification
Step-II) Retrotranscription
Step-III) PCR
Step-IV) Agarose gel electrophoresis
What is the expected length of the PCR product?
The PCR product is analyzed on agarose gel with the expected length of 313 bp.
How to calculate RNA Quantification?
• The results of absorbance at 260nm and 280nm are 0.0743 and 0.0416, respectively.
• The RNA purity result by 260/280 ratio is 1.786.
• The RNA concentration is 293.6 μg/ml with respect to optical density at 260nm. Which is calculated as follows:
1 OD= 40 μg/ml of RNA
0.0743= X μg/ml => X= 293.6 μg/ml
How to calculate the amount of RNA that will be used in the retrotranscription process?
The amount of RNA that will be used in retrotranscription should be 1.0 μg and it is calculated as follows:
m1V1=m2V2
(293.6 μg/ml)(1000μl)=(1μg)(X μl)
X=3.406 μl
How to calculate the amount of Ethidium Bromide that will be added to the agarose gel mixture?
The amount of Ethidium Bromide that will be added into agarose gel mixture is calculated as follows:
The stock of Ethidium Bromide is 10mg/ml, and the needed concentration is 1μg/ml.
m1V1=m2V2
(10mg)(X)=( 1μg)(150ml)
X=15μl
What is the importance of RNA Quantification?
RNA quantification is an important part for the experiment because the result shows the purity of the RNA and calculations that are made by using it are required for the rest of the experiment.
Nucleic acids are absorbed at 260nm while other components are absorbed at 280nm.
Therefore, the ratio between the absorbances at 260nm and 280nm should be in the range of 1.6-2.0: if the result is closer to 2.0 it is purer but if it is lower than 1.6 it shows that there is the presence of protein or other contaminants.
How RNA concentration is obtained?
The calculation of RNA concentration is obtained with respect to optical density at 260nm by taking 1 unit of optical density as 40 μg/ml of RNA.
Thus, the RNA concentration is 293.6 μg/ml.
This concentration determines the amount of RNA that will be used in retrotranscription protocol where 1.0 μg of RNA is needed.
So, the volume of the RNA will be 3.406 μl.
If the concentration is higher, calculations would be different, and the amount of water should be reduced accordingly.
What can be the reason for obtaining different results in the gel electrophoresis?
When the image of gel electrophoresis is analyzed, there are 3 wells for each student and they are in order of +RT, -RT and NTC.
There is 3 different scenarios:
1) there is a band in Well-2 and it looks same with Well-1 which is the expected result for +RT, but not for -RT. There shouldn’t be any band both for -RT and NTC if the experiment is done correctly.
There can be several possible explanations for this result: either the student put reverse transcriptase (RT) enzyme instead of water in the retrotrancription step or the student load Well-1 and Well-2 with the same sample which in this case it is +RT.
2) there is a slight band in the Well-5 which is the -RT well for that student.
The reason for this faded band is contamination. Probably, the student took some of the interphase and organic phase while recovering the aqueous phase containing RNA.
To prevent this to happen, smaller and thinner pipette tip can be used, and recovery process can be done in a couple times, instead of one big volume recovery.
3) Other results are correct and what is expected to be seen.
Why RT-PCR is used?
Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a technique used for obtaining complementary DNA (cDNA) from RNA by reverse transcriptase enzyme and amplifying it for further studies.
In this experiment, it is used to detection of GAPDH gene expression in HeLa cells.
Reporter genes: description and application
A reporter gene is a gene which activity can be easily monitored. It encodes for a genetic product that can be used to study the activity of some regulated sequences of another gene. We use this method to evaluate the gene and promoter expression (such as in the experiment done by us). A reporter gene is a gene that does not have a similar function of the gene of interest and his reporter activity must not interfere with physiological processes: the signaling pathways and cell metabolism must not be affected. In general, a reporter gene has to avoid cellular toxicity, has an high rate of turnover and it emits a wavelength (important for in vivo analysis and in vivo imaging). Moreover, the reporter activity assay must be specific, reproducible and not time-consuming. Some examples of reported genes are:
• GFP (green fluorescent protein)→ It is a protein that has multiple variants and different excitation and emission spectra. It was extracted from Jellyfish and modified to be used in human. GFP is not bioluminescent, is pure excitation and it emits green fluorescence under UV excitation. This is allows thanks to the chromophore inside, formed by 3 aa (serine, threonine and glycine). GFP is composed by 11 anti-parallel beta strands that form a cylinder and inside there is an alpha-helix and the chromophore (fluorescent core). GFP has a lot of different variant based on the color of emission: YFP, CFP, E-GFP, BFP, UV-GFP… For example, if you fuse GFP to your protein of interest, you simply use different variants to check where the protein goes in the cell. You can monitor molecular dynamicity thanks to the fluorescence emitted by the GFP linked with the protein of interest. It can be useful to study the localization of specific protein or receptor within the cells.
• Chloramphenicol acetyl-transferase. This is a bacterial enzyme that is able to acetylate chloramphenicol, used in the mammalian cells as a reporter gene. We can do an enzymatic test. Usually you take chloramphenicol that has been labelled with C14 and you add this in the presence of the extract from your cells. The Chloramphenicol acetyl-transferase present in your extract can acetylate the chloramphenicol that has been labelled with the isotope C14. Then you perform a thin layer chromatography, where you put your sample. The sample runs in the presence of a solvent that allows the chloramphenicol to move on the surface. From the bottom to the top of the layer you will have in some case, some of the chloramphenicol not acetylated, and then you start to have the acetylated chloramphenicol. The signal that you get from the acetylated chloramphenicol is a measure of the presence of chloramphenicol in your extract.
• β-galactosidase, codified by LacZ gene of E. coli. The enzymatic reaction is the conversion of the substrate in colored compounds. You measure the appearing of a yellow solution that comes from the degradation of this compound: degradation of ONPG (o-nitrophenyl-b-D-galactopyranoside) → yellow soluble compound coming from your transfected cells.
• Luciferase (used by us). We can perform an enzymatic test based on the oxidation of his specific substrate (luciferine in the case of FireFly Luciferase and coelenterazine in the case of Renilla Luciferase), where the product that we measure in this case is light. The different kinds of Luciferase are expressed in different animals, such as bacteria, marin invertebrates (in particular FireFly Luciferare is expressed in the fireflys)… but not in mammals.
Reporters genes are used for a variety of applications: to study gene expression in the case of transcription and post-transcriptional regulation or post-translational regulation (protein localization, stability and interactions), but they are used also to monitor cellular events such as receptor activation and signaling, pathway analysis (such as protein-protein interaction) and in case of disease or immune response, for example in case of infection or drug therapy.
quantitative PCR and its application: pros and cons of TaqMan and SYBR Green based chemistry
Quantitative PCR, or real-time PCR, (qPCR) uses the linearity of DNA amplification to determine absolute or relative quantities of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation in the qPCR assay. In qPCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the threshold cycle (CT) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against CT. The amount of DNA or cDNA in an unknown sample can then be calculated from its CT value. Main quantitative PCR applications: • Quantitation of gene expression • siRNA experiments • Microarray verification • CNV • Genotyping • microRNA analysis • infection disease/pathogen detection • viral quantification • SNP genotyping • Quality control and assay validation • Drug target validation • Food safety testing • GMO detection • Genotype alleles in divergent individuals of the population • Other applications During PCR process, there is a curve of amplification and an exponential phase. Usually, the value you get is the cycle that goes above the threshold line. You can compare this Cq value (when the signal goes over the threshold) of several curves for several genes. TaqMan and SYBR Green based chemistry are two important methods to evaluate the amount of DNA amplified by Quantitative Real Time PCR. In fact, there’s a direct proportion between the fluorescent signal that they emit and the amplified DNA. SYBR Green intercalates in the double strand DNA (in particular it anneals in the minor groove of DNA), once it forms and it emits fluorescence that we can detect. Instead the TaqMan chemistry is based on a specific probe, an oligonucleotide that has at the 5’ end a fluorophore and at the 3’ end a quencher. This probe is complementary to the DNA filament we want to amplify, but when it is intact, the fluorophore is not able to emit fluorescence due to the presence of the quencher. So, it anneals to one of the two strands and when the polymerase starts to amplify the DNA, it reaches the probe and it degrades it, releasing the fluorophore. Once far away from the quencher, the fluorophore starts to emit its fluorescence, that we can detect. The pros of SYBR Green is that it is cheaper than TaqMan probe, but it has a long and difficult setting. Instead TaqMan is more expensive, but it has a particular advantage. In fact if we’re performing a RT-PCR we can draw a specific probe that is able to bind to the exon-exon splicing junction of the cDNA we want to amplify and in this way we avoid the amplification of unspecific DNA eventually left in the sample (or an intron among the exons). This kind of probe, that bind the exon-exon junction, is able to say me if there are genomic contamination among the exons. In both of them we need to add the forward and the reverse primers. SYBR green is an aspecific method because it can not say us if there is an external genome that has contaminated the specific sequence which I am interested in or if there are some introns among the exons. But thanks to this method, we are able to know if some dimer of primers were formed, because thanks to a graph that the software of analysis creates, we can observe the value of the melting temperature. If it has a lower peak on the left, compared to a normal graph, it means that some dimers of primers were formed in the sample.
methods for mammalian cell transfection;
The method to transfect mammalian cells must be highly efficient, with low toxicity and it must be the right one for the cell line used, it must be reproducible in vitro and in vivo. The problem is to overcome the natural bias of the cell line, such as the polarity of DNA and the lipophilic cell membrane. Transfection starts with the introduction of DNA into cells, then continues with the expression of the gene, the selection of stably transfected cells and the characterization of the produced protein. We can use two different kinds of methods: chemical and physical.
CHEMICAL METHODS:
• Calcium phosphate → It can be used for many cell types, You simply have a solution with calcium phosphate and the DNA. DNA in a solution of calcium phosphate creates a precipitate in the cells. The advantage is that this method is very cheap, you have a solution and you don’t need to buy anything else. However, it carries some technical difficulties, for example it is difficult to create homogeneous precipitates, you should see like a “sand” in your cells, but you often obtain complexes of different sizes. Also, it is not very efficient, it could be very toxic for some cells, not all cell lines can be transfected with this method and this method is affected by minimal variation of pH.
• Liposomes (lipofection) → This is the one we used in the lab. Liposomes are a mixture of polycationic and neutral lipids that form a unilamellar liposome vesicle with a positive net charge. This charge can interact with the negative charge of DNA, then DNA-lipid complexes fuse with the cell membrane and the content is released inside the cell. The advantage is the low toxicity, but it depends on the cell line and the mixture you use: there are many different mixtures commercially available. The disadvantages are that it is difficult to produce the liposomes. It depends on the operator, for this reason, there may be very high variability. In laboratory, we used FUGENE-HD that is a cationic lipids-based reagent to transfect DNA into mammalian cells. It’s simple and efficient, with low toxicity compared to the LIPOFECTAMINE. This is the process: you have the liposome complexed to the DNA, they enter into the cell, some will be degraded by the endosomal department and part will go to the nucleus and express the gene.
PHYSICAL METHODS:
• Electroporation → it relays on a short electrical pulse. You have two channels, cells get together with the DNA, you apply an electric field and the DNA enters into the cells. This method is very toxic: you have to use lots of cells because half of them will die. It is difficult to setup the conditions and optimize the intensity and duration of the electric pulse, but sometimes it is the only method that can be used for some cell lines. DNA enters directly without passing by the endosomal compartment where it can be degraded.
• Microinjection → DNA is inoculated into the cell (in the cytoplasm or in the nucleus) by a glass capillary, under high pressure. Due to the reduced size of the cells, microinjection requires a high sophisticated apparatus that include: a microscope, a micromanipulator and an injector. Using a microscope with glass capillaries, you can transfect one cell at a time injecting the DNA directly inside the nucleus. It is possible to use microinjection in stem cells to produce OGM. The advantages of this technique are: the transfer of DNA directly into the cytoplasm or into the nucleus, the expression efficiency increase and the expression level can be easily modulated. While the disadvantages are: it is technically difficult, can be done few cells at a time and it is expensive.
• Gene gun → Gold particles covered with DNA are shot directly into the cells or tissue, under high pressure. Usually for tissues because it is not very easy to shoot the cells. Having a gun is quite expensive. One important thing is that you have to optimize the process of transfection: the DNA must be cleaned, and you have to choose the correct plasmid vector. The transfection time must be precise, for example it is better not to leave the reagents for long periods because they can be very toxic. The advantages are: it is an in situ transfection, is feasible for cell system hard to transfect with other methods, especially in vivo, gene expression studies under normal physiological conditions are permitted: tissue specific expression and development and less DNA and less cells can be used. The disadvantages are: it is an invasive technique, efficiency strictly dependent on cell type, there are many parameters to control and it is expensive.
mechanisms for studying gene expression
primary and secondary cultures: pros and cons
relevant parameters in setting up a PCR experiment and its application
