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