DNA & RNA extraction

• Basic nucleic acid extraction methods are covered
• Focus is on overview, DNA and RNA extraction methods, and quality and quanitity analysis

Nucleic Acid Extraction

• Involves releasing nucleic acids from cells
• Results should be free of contaminants like proteins, carbohydrates, and lipids
• This is a preliminary step in DNA and RNA studies
• Multiple methods and commercial kits are available
• Nucleic acids can be isolated from almost any intact cellular tissue
• Optimization is needed depending on the sample source

Choosing a Method

• Consider the efficiency and cost-effectiveness of extraction
• Ensure sufficient quantity of extracted material for downstream applications
• Prioritize the purity of the final nucleic acid extract

Steps

• Isolation of nucleic acid
• Purification of the sample
• Concentration of the sample

Uses

• Used for DNA/RNA studies in disease molecular biology and biotechnology research
• Can identify etiology of infections and resistance to drugs
• Used to predict disease progression
• Used in paternity studies and suspect identification in forensics

DNA Extraction Steps

• Pretreatment and weighing may be necessary
• Weighing allows for final DNA yield calculation later
• Cell lysis is next
• Outer boundaries are disrupted to release intracellular components, either physically or with solutions

Physical Cell Lysis

• Physical cell lysis methods include grinding, shearing, bead beating, and freeze-thaw
• This method is efficient for a wide range of cells
• Offers control over buffer use, eliminating potential contaminants
• High lysing efficiency
• Requires equipment
• Reproducibility may vary
• Not compatible with small volumes
• Protein denaturation/aggregation can occur
• Subcellular components may be subjected to disruptive forces

Chemical Cell Lysis

• Relies on alkali like NaOH
• Also relies on detergents like SDS and CTAB
• Chaotropic agents like EDTA are also used
• Is a rapid, gentle, efficient, and reproducible method
• Can extract total protein/subcellular fractions from samples
• Is easily adaptable for small sample volumes or higher throughput
• Can be used with mechanical methods for complete cell disruption
• Some buffer components may need to be removed
• High salt and detergent concentrations are incompatible with some protein assays and mass spectrometry
• Works well with cultured cells but may not be effective for some tissues

Removal of Contaminants

• Lipids are addressed by adding detergent
• This results in the formation of micelles surrounding hydrophobic lipid molecules
• Examples are SDS and CTAB
• Proteins are removed using proteases and detergents
• Proteinase K is a broad-spectrum serine protease that degrades nucleases
• SDS makes proteins anionic
• RNA can be removed with RNAse to create RNA-free samples

DNA Precipitation

• It separates free DNA from cellular debris
• Organic, inorganic, solid-phase methods are used

Organic DNA Precipitation

• Uses high salt concentrations in low pH, using 1:1 phenol-chloroform
• Formation of biphasic emulsion is centrifuged
• The supernatant with DNA is placed into a new tube for ethanol precipitation using ice-cold reagents in a low-temperature environment
• Solvation shell clusters solvent molecules surrounding/attaching to solute molecules in solution
• Effective
• Reliable
• Works with a wide range of samples
• Time-consuming
• Uses hazardous reagents
• Leads to multiple tube transfers

Inorganic DNA Precipitation

• Also called "salting out"
• Selectively precipitates proteins while leaving DNA in solution using low pH and high salt conditions
• DNA is precipitated as in organic methods, using isopropanol
• Faster and easier than other methods
• Uses non-toxic chemicals, so is safer
• Involves fewer steps that could introduce contaminants
• Inadequate removal of salts can affect DNA mobility during electrophoresis, causing band shifting

Solid-Phase DNA Precipitation

• Solid matrices bind and wash DNA
• These tubes are solid phase columns
• Columns come in various sizes based on needing to isolate more or less DNA
• Examples are silica-based, magnetic, and anion exchange
• Is a quick, reliable method
• Produces high-quality DNA
• Usually sold as a kit
• Can be expensive

DNA Resuspension

• Involves use of TE buffer so the extract can be stored/diluted
• EDTA chelates divalent ions that could potentially form salts with phosphate groups of DNA
• Also inhibits DNAses
• Basic pH reduces interactions between DNA, histones, and polycationic amines

RNA Extraction

• RNA is less stable than DNA
• RNAse are ubiquitously present in the environment
• While dissociating tissue, samples should be frozen or put in a buffer, to inactivate intracellular RNAse

Organic RNA Extraction

• Cell lysis is done with detergent or phenol and high salt (0.2-0.5 M NaCl) or RNAse inhibitors, DNAse may be added
• Involves separating proteins by acid phenol:chloroform:isoamyl alcohol
• Involves separating of phases by TRIzol reagent
• RNA is precipitated by 2x ethanol or 1x isopropanol
• RNA is resuspended in nuclease-free water
• Rapidly eliminates nucleases
• Stabilizes RNA
• Can be used for smaller or larger samples
• Protocols are well-established and more routinely used
• Can be time-consuming and laborious
• Makes use of hazardous reagents

Solid-Phase RNA Extraction

• Cell lysis similar to organic extraction
• Lysate is added to the spin column with high-salt chaotropic buffers in microfuge tubes
• RNA is washed and eluted from the membrane
• Simple and straightforward
• Sold as kits
• Usable in large-scale extractions and automated methods
• Large amounts of sample cannot be used in one go because they can clog the membranes
• Can be expensive

Measurement of Quantity and Quality

• Assessed to check concentration and purity of yield
• Also determines the success of isolation
• Used for determining calculations for downstream applications

Methods

• Spectrophotometry is used
• Agarose gel electrophoresis is also used

Spectrophotometry

• Follows Beer-Lambert's Law to determine concentration of DNA
• Nucleic acids have conjugated double bonds in purine and pyrimidine rings
• Maximum absorbance observed at 260 nm
• 1 absorbance unit at 260 nm is about 50 μg/mL of DNA
• About 40 μg/mL of RNA
• Nanodrop spectrophotometry requires only 1–2 μL of sample instead of the standard 50-75 μL
• Displays the entire absorbance spectrum in a graphical form
• Can determine a wide range of sample concentrations without serial dilution (2 ng–15,000 ng/μL)

Purity Assessment

• A260/A280 ratio is used to assess purity
• Check absorbance at 260 nm for nucleic acids
• Check absorbance at 280 nm for proteins
• Check additional A230 nm tests for contaminants and 270 nm for phenols
• Optimal A260/A230 and A260/A280 ratio are between 2.0–2.2 and 1.8-2.00 for RNA
• For DNA
• 2.0–2.2 and 1.8-2.00

Agarose Gel Electrophoresis (AGE)

• It is a separation method based on size under the influence of an electric current (max: 15V)
• After running, bands of interest can be isolated and re-extracted for further studies
• Need to plot log of observed molecular weight based on the marker against distance traveled by each of the bands