Successful DNA and RNA extraction – technologies,

techniques and troubleshooting24 May 2018

Did you know…

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• Each one of your cells contains about 2-3m of DNA

• If you unravelled the DNA from all your cells it would stretch to the moon over 300,000 times, 10 billion miles

• All digital information in this world can be stored on 2 grams of DNA

• It would take 50 years to type the entire human genome if someone types at a speed of 60 wpm and works 8 hours a day!

• The first isolation of DNA was performed in 1869 by Friedrich Miescher, a Swiss physician and biologist

• It was only in 1943 that scientists became aware of the fact that genetic information is stored in DNA, not in proteins

What is DNA?

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Commonality

50%

99.9%96%

60%

80%

70%

Why extract/study DNA/RNA?

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• Research & Development

• Diagnosing disease and genetic disorders

• Crime identification, OJ Simpson trial

• Historical identification - DNA has been used to identify September 11th victims and casualties of the Jewish Holocaust

• Identify certain origins of wine by tracing the lineage of various grapevines

• Tracing decreasing populations of various species, including the Grizzly Bear

• DNA testing is used to authenticate food like caviar

Start with the end in mind…

• The first step in any study investigating genetic differences in an organism of interest and all other genomic experiments is the purification of pure nucleic acids

• Often thought of as insignificant part of processes - the importance of high-quality, purified DNA/RNA cannot be underestimated

• Questions to ask before deciding on extraction method:• What are you going to be using the

DNA/RNA for?

• Do you have limited starting material?

• How long has your sample been stored?

• What are your timelines, do you need rapid TAT?

• Do you have a budget?

• Is your sample high risk?

• Regulatory requirements?

Basic DNA Extraction

Lysis - this is often done by sonicating or bead beating the sample. Vortexing with phenol is often effective for breaking down proteinaceous cellular walls or viral capsids. The addition of a detergent such as SDS is often necessary to remove lipid membranes.

Protein Removal - DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform and centrifuged the proteins will remain in the organic phase and can be drawn off carefully. The DNA will be found at the interface between the two phases.

Precipitation - DNA is precipitated by mixing with cold ethanol or isopropanol and then centrifuging. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol wash removes the salt previously added.

• Removal of DNA from the cells or viruses in which it normally resides

RNA Extraction

• Removal of RNA from biological samples

• Complicated by presence of ribonuclease enzymes (RNases) in cells and tissues which can rapidly degrade RNA

Extraction Methods

DNA

• Organic

• Silica column based

• Magnetic bead based

• In tip (TRUtip)

RNA

• Organic

• Silica column based

• Magnetic bead based

• Direct lysis

• In tip (TRUtip)

Organic Nucleon Chemistry

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Advantages

• Wide variety of different sample types, blood, tissue, saliva,

cells, swabs

• High purity DNA

• Recover in-process lost DNA easily

• Scalable

• Good for long term Biobanking

• Clotted samples, aged blood

Disadvantages

• Throughputs limited, labour intensive

• The use and associated waste of chlorinated organic reagents

• Difficult to automate

• Gene Expression

• Invader

• Bioinformatics

Silica Column Based

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Qiagen kits, PureLink™ kits, NucleoSpin® kits

Advantages

• Easy to use, off shelf solutions, high purity DNA/RNA

• Wide variety of kits available for different sample types,

blood, tissue, saliva

• Automatable – some kits

Disadvantages

• Fixed binding capacity, limited input amounts, filters clog

• Throughputs limited, labour intensive

• Limited by elution volumes

• Expensive

• Short expiry dates

• Gene Expression

• Invader

• Bioinformatics

Magnetic Bead Based

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Advantages

• Easy to use, off shelf solutions RNA/DNA

• No risk of filter clogging

• Rapid TAT

• Wide variety of kits available for different

sample types, blood, tissue, saliva

• Automatable

• Gene Expression

• Invader

• Bioinformatics

Disadvantages

• Often require specific compatible platforms

• Limited by input amounts / elution volumes

• Laborious capture/release beads if manual

• Expensive

• Bead carryover in DNA/RNA

• Improper distribution of beads, slow in

viscous solutions

Chemagen kits, Norgen kits, MagMax kits, GeneCatcher™

Direct Lysis

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Advantages

• Extremely fast and easy

• DNA and RNA from range of sample types

• Works well with small samples

• Amenable to simple automation

• Scalable

• Gene Expression

• Invader

• Bioinformatics

Disadvantages

• Inability to perform traditional analytical methods such as spectrophotometric measurement of yield

• Dilution-based (most useful with concentrated samples)

• Potential for suboptimal performance unless developed/optimized with downstream analysis

• Potential for residual RNase activity if lysates are not handled properly

Isohelix kits, Ambion kits (Thermo)

Lysis buffer formulations to disrupt samples, stabilise DNA/RNA

In tip

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Akonni TruTip

Salt chemistry, glass capture matrix in pipette tip

Advantages

• DNA and RNA from range of sample types

• Simple, fast, cheap

• Easily automatable

• Can use with viscous samples – large porosity

• Gene Expression

• Invader

• Bioinformatics

Disadvantages

• Requires upfront homogenization and cell disruption step

• Specific benchtop instrument required

• Limited by input amounts

• Limited by elution volumes

Nucleon vs Others

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Chemistry Company Blood VolRange

Elution/Rehydration

Volume

Total Max Yield/10mLblood (µg)

Tepnel Nucleon™Organic Tepnel 0.3-50mL any <800µg

QIAamp DNA Blood Maxi Kit Silica Qiagen 3-10mL 500-2000µL <600µg

Wizard® Genomic DNA Kit Organic Promega 0.3-10mL 800µL <500µg

GeneCatcher™Magnetic Bead Life

Technologies 3-10mL 1mL <300µg

Gentra PureGeneBlood Kit Organic Qiagen 0.3-30mL 250-1000µL <350µg

NucleoSpin® Blood XL Silica MACHEREY-

NAGEL 2-10mL 600-2000µL <300µg

Tru TipIn tip Akonni 0.1-2mL 50-100µL <250µg

Throughputs

• Manual vs semi-automated vs fully-automated?• Labour availability / lab capacity

• Sample numbers

• Throughput time required

• Material cost per sample

• Technically challenging – trained staff available?

• Hazardous chemicals (phenol, chloroform)

Common Issues• Low concentration DNA/RNA

• Low sample input

• Inefficient lysis or protein degradation

• Low % ethanol used instead of 100%

• Rehydration volume too high

• Change extraction method or concentrate up afterwards using centrifugal micro concentrators

• Low quality DNA/RNA (pure DNA = 1.8, pure RNA = 2.0)• Low A260/A280 ratio - inefficient lysis or protein degradation, contamination by

residual phenol, guanidine or other extraction reagent

• High A260/A280 ratio - High level of residual RNA/DNA, pure quality blank

• https://tools.thermofisher.com/content/sfs/brochures/T123-NanoDrop-Lite-Interpretation-of-Nucleic-

Acid-260-280-Ratios.pdf

Common Issues

• DNA/RNA does not perform well in subsequent enzymatic reactions• Not enough DNA/RNA in sample

• Inhibitory substances in preparation

• High level of residual RNA/DNA

• Limited amount of material can be loaded onto column

• Blocked filters/membranes

• Clotted/aged blood – digest with PK prior to extracting

Common Issues - RNA• Degraded RNA

• Sample collection not optimal - freeze/protect samples at point of collection

• Sample thawed during transit

• Extracted RNA undergone too many freeze-thaw cycles. Store at -80oC.

• Work on ice, pre-chill buffers

• RNases introduced during extraction• Equipment used needs to be thoroughly cleaned and kept separate from

common lab equipment, for e.g. RNase ZAP and DEPC water

• Wear gloves, bare hands are a source of RNase contamination. Changes gloves frequently.

• Don’t use opened boxes of tubes/tips

• Use RNase-free water and reagents

• Keep tube lids closed whenever possible

Complicated Samples

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• Archaeological samples or FFPE samples containing partially

degraded DNA/RNA

• Samples containing inhibitors of subsequent analysis procedures,

most notably inhibitors of PCR, such as haemoglobin in blood

• Samples from microorganisms or plants with thick cellular wall, for

e.g. yeast

• Aged samples such as blood stored long term

Sample Types

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Extraction of DNA, RNA and miRNA from multiple sample types:

Whole Blood

Lymphocytes

Buffycoats

Plasma

Serum

PBMCs

Granulocytes

Lymphocytes

Leukocytes

Bloodspots

Bone marrow

Saliva

Buccal scrapes

Swabs and mouthwash samples

Cells / cell pellets

Biopsies

FFPE

Fresh frozen tissue

Fat

Bull semen

Otter spraint

Urine

Ductal lavage

Pancreatic juice

Feathers

Corneas

One last thought…

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Orbiting our Earth is a memory device that is known as ‘Immortal Drive’. The device is actually inside the International Space Station and it contains digitalized DNA sequences of Lance Armstrong, Stephen Colbert, Stephen Hawking and others. It is actually an attempt to preserve human race in event of a global catastrophe.