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