Chapter 3

Nucleic Acid Extraction Methods

Outline MEASUREMENT OF NUCLEIC ACID QUALITY AND QUANTITY

Electrophoresis
ISOLATION OF DNA Spectrophotometry
Preparing the Sample Fluorometry
Bacteria and Fungi Microfluidics
Viruses
Nucleated Cells in Suspension (Blood and Bone Marrow
Aspirates)
Plasma
Tissue Samples
DNA Isolation Chemistries Objectives
Organic Isolation Methods
Inorganic Isolation Methods 3.1 Compare and contrast organic, inorganic, and
Solid-Phase Isolation solid-phase approaches for isolating cellular and
Proteolytic Lysis of Fixed Material mitochondrial DNA.
Rapid Extraction Methods 3.2 Describe DNA isolation from minimal and
Isolation of Mitochondrial DNA challenging samples.
ISOLATION OF RNA 3.3 Compare and contrast organic and solid-phase
Total RNA approaches for isolating total RNA.
Specimen Collection 3.4 Distinguish between the isolation of total RNA and
RNA Isolation Chemistries that of messenger RNA.
Organic Isolation 3.5 Describe the gel-based, spectrophotometric, and
Solid-Phase Isolation fluorometric methods used to determine the
Proteolytic Lysis of Fixed Material quantity and quality of DNA and RNA preparations.
Isolation of polyA (Messenger) RNA 3.6 Calculate the concentration and yield of DNA and
RNA from a given nucleic acid preparation.

78
 Chapter 3 • Nucleic Acid Extraction Methods 79

The purpose of nucleic acid extraction is to release the

nucleic acid from the cell for use in subsequent proce- TABLE 3.1 Yield of DNA From Different
dures. Ideally, the target nucleic acid should be free of Specimen Sources29-35
contamination with protein, carbohydrate, lipids, or other
nucleic acids, that is, DNA free of RNA or RNA free Expected
Specimen Yield*
of DNA. The initial release of the cellular material is
achieved by breaking the cell wall (if present) and cell Specimens Adequate for Analysis Without DNA Amplification
and nuclear membranes (cell lysis). Lysis must take place
in conditions that will not damage the nucleic acid. Fol- Blood† (1 mL, 3.5–10  × 106 WBCs/mL) Buffy 50–200  μg
coat† (1 mL whole blood)
lowing lysis, the target material is purified, and then the
concentration and purity of the sample can be determined. Bone marrow† (1 mL) 100–500  μg

Cultured cells (107 cells) 30–70  μg

ISOLATION OF DNA Solid tissue‡ (1 mg) 1–10  μg

Lavage fluids (10 mL) 2–250  μg

Although Miescher first isolated DNA from human cells
in 1869 by precipitation,1 the early routine laboratory Mitochondria (10-mg tissue, 10 cells) 7
1–10  μg
procedures for DNA isolation were developed from
density-gradient centrifugation strategies. Meselson and Plasmid DNA, bacterial culture (100-mL 350  μg–1 mg
overnight culture)
Stahl used such a method in 1958 to demonstrate semi-
conservative replication of DNA.2 Later procedures took Bacterial culture (0.5 mL, 0.7 absorbance 10–35  μg
advantage of solubility differences among chromosomal units)
DNA, plasmids, and proteins in alkaline buffers. Large Feces§ (1 mg; bacteria, fungi) 2–228  μg
(greater than 50 kbp) chromosomal DNA and proteins
cannot renature properly when neutralized in acetate at Specimens Adequate for Analysis With DNA Amplification
low pH after alkaline treatment, forming large aggregates Serum, plasma, cerebrospinal fluid|| (0.5 mL) 0.3–3  μg
instead. As a result, they precipitate out of solution. The
relatively small plasmids return to their supercoiled state Dried blood (0.5- to 1-cm-diameter spot) 0.04–0.7  μg
and stay in solution. Alkaline lysis procedures were used
Saliva (1 mL) 5–15  μg
extensively for extraction of 1- to 50-kb plasmid DNA
from bacteria during the early days of recombinant DNA Buccal cells (1 mg) 1–10  μg
technology.
Bone, teeth (500 mg) 30–50  μg

Hair follicles# 0.1–0.2  μg

Preparing the Sample
Fixed tissue** (5–10  × 10-micron sections; 6–50  μ
Nucleic acid is routinely isolated from human, fungal, 10 mm2)
bacterial, and viral sources in the clinical laboratory
(Table 3.1). The initial steps in nucleic acid isolation Feces†† (animal cells, 1 mg) 2–100 pg
depend on the nature of the starting material and the test
method. Some test systems include sample-collection *Yields are based on optimal conditions. Assays will vary in yield and purity
of sample DNA.
vessels and reagents that begin the nucleic isolation in †
DNA yield will vary with WBC count.
transport to the laboratory. Details of sample collection ‡
DNA yield will depend on type and condition of tissue.
§
are important because of the diversity of sample types ||
Different bacterial types and fungi will yield more or less DNA.
DNA yield will depend on degree of cellularity.
and nucleic acid sources (viruses, bacteria, nucleated ¶
Dried blood yield from paper is less than from textiles.
cells). Use of an improper collection tube or method #
Mitochondrial DNA is attainable from hair shafts.
will compromise the test results. Test validation should **Isolation of DNA from fixed tissue is affected by the type of fixative used
and the age and the preliminary handling of the original specimen.
include the sample-collection methods. ††
Cells in fecal specimens are subjected to digestion and degradation.
80 Section II • Common Techniques in Molecular Biology

Viruses
Advanced Concepts
Viral DNA is held within free viruses or integrated into
In surveying the literature, especially early ref- the host genome along with host DNA. Some proce-
erences, the starting material determined which dures use cell-free specimens, such as plasma, for viral
DNA extraction procedure was used. Extraction detection. Others may require concentration of viroids
procedures were also modified to optimize the by centrifugation or other methods.
yield of specific products. A procedure designed
to yield plasmid DNA did not efficiently isolate Nucleated Cells in Suspension (Blood and Bone
chromosomal DNA and vice versa. Marrow Aspirates)
Nucleic acid in human blood or bone marrow comes
mostly from white blood cells (WBCs). Anticoagulants
Bacteria and Fungi added to the collected sample will prevent clotting,
which will trap the WBCs. Serum from clotted blood
Many of the early recombinant DNA experiments were is a rich source of proteins, lipids, and other molecules,
performed with gram-negative bacteria. Methods used at but not nucleic acids. Free WBCs carrying nucleic acids
that time are the basis for a variety of DNA isolation and cell-free nucleic acids are available from plasma.
systems currently used. Toxic substances used in the WBCs in blood or bone marrow specimens are purified
older methods have been replaced with safer materials. of red blood cells (RBCs) and other blood components
Cell walls are not thick and can be lysed by high pH and by either differential density-gradient centrifugation or
detergents. differential lysis.
Some bacteria and fungi have tough cell walls that For differential density-gradient centrifugation,
must be broken to allow the release of nucleic acid. whole blood or bone marrow mixed with isotonic saline
Several enzyme products that digest cell wall polymers is overlaid with Ficoll. Ficoll is a highly branched
are commercially available. Alternatively, cell walls can sucrose polymer that does not penetrate biological
be broken mechanically by grinding or by vigorously membranes. Upon centrifugation, the mononuclear
mixing with glass beads. Gentler enzymatic methods WBCs (the desired cells for isolation of nucleic acid)
are less likely to damage chromosomal DNA and thus settle into a layer in the Ficoll gradient that is below
are preferred for methods involving larger chromosomal the less dense plasma components and above the poly-
targets as opposed to plasmid DNA. Treatment with morphonuclear cells and RBCs. The layer containing
detergent (1% sodium dodecyl sulfate) and strong base the mononuclear WBCs is removed from the tube and
(0.2 M NaOH) in the presence of Tris base, ethylene- washed by rounds of resuspension and centrifugation in
diaminetetraacetic acid (EDTA), and glucose can also saline before proceeding with the nucleic acid isolation
break bacterial cell walls. procedure.
Boiling in dilute sucrose, Triton X-100 detergent, Another method used to isolate nucleated cells takes
Tris buffer, and EDTA after lysozyme treatment releases advantage of the differential osmotic fragility of RBCs
DNA that can be immediately precipitated with alcohol and WBCs. Incubation of whole blood or bone marrow
(see following discussion). DNA extracted by boiling in hypotonic buffer or water will result in the lysis of
or alkaline (NaOH) procedures is denatured (single the RBCs before the WBCs. The WBCs are pelleted
stranded) and may not be suitable for methods, such as by centrifugation, leaving the empty RBC membranes
restriction enzyme analysis, that require double-stranded (ghosts) and hemoglobin, respectively, in suspension
DNA. and solution.
Commercial reagents designed for isolation of DNA
in amplification procedures, such as polymerase chain
Plasma
reaction (PCR), are used for yeast, filamentous fungi,
and gram-positive bacteria. The advantage of these types Solid tumors and transplanted organs release cells, exo-
of extraction is their speed and simplicity. somes, and nucleic acid into the bloodstream.3,4 Shed
 Chapter 3 • Nucleic Acid Extraction Methods 81

cells have the molecular characteristics of the shed-

ding tissue or organ, as does cell-free nucleic acid. TABLE 3.2 Tissue Fixatives Influencing
Exosomes are small vesicles (30 to 100 nm in diame- Nucleic Acid Quality7
ter), which form by invagination and budding from the
inside of cellular endosome vesicles and are secreted by Relative Average Fragment
Quality of Fixative Size
living cells. They contain proteins, lipids, and nucleic Fixative Nucleic Acid Range (kb)
acid.5 They can be collected by centrifugation. With
the high sensitivity achievable from amplification pro- 10% buffered Good 2.0–5.0
cedures, these sources of circulating nucleic acids can neutral formalin
be detected for purposes of diagnostic and prognostic Acetone Good 2.0–5.0
analyses. Such a test is called a liquid biopsy. Liquid
biopsies may preclude surgical biopsies and allow Zamboni’s Not as good 0.2–2.0
serial biopsy testing. Isolation of cell-free nucleic acid Clarke’s Not as good 0.8–1.0
requires procedures to concentrate the target nucleic
acid before isolation. These procedures include solid- Paraformaldehyde Not as good 0.2–5.0
phase collection of nucleic acid on beads or columns
Methacarn Not as good 0.7–1.5
(see following discussion). Circulating tumor cells can
be collected on special columns and filters that will Formalin–alcohol– Not as good 1.0–4.0
selectively bind to tumor cells, avoiding WBCs that may acetic acid
be present. B-5 Less desirable <0.1

Carnoy’s Less desirable 0.7–1.5

Tissue Samples
Zenker ’s Less desirable 0.7–1.5
Fresh or frozen tissue samples are dissociated before
DNA isolation procedures can be started. Grinding Bouin’s Less desirable <0.1
the frozen tissue in liquid nitrogen, homogenizing the
tissue, or simply mincing the tissue using a scalpel
disrupts the whole-tissue samples. Fixed, embedded
tissue may be deparaffinized by soaking in xylene DNA Isolation Chemistries
(a mixture of three isomers of dimethylbenzene). Less
Organic Isolation Methods
toxic xylene substitutes are also often used for this
purpose. After xylene treatment, the tissue is rehydrated After release of DNA from the cell, further purification
by soaking it in decreasing concentrations of ethanol. requires removal of contaminating proteins, lipids, car-
Alternatively, fixed tissue may be used directly without bohydrates, and cell debris. For organic isolation, this is
dewaxing. accomplished using a combination of high salt, low pH,
Depending on the fixative, DNA in preserved tissue and an organic mixture of phenol and chloroform. The
may be broken and/or cross-linked to varying extents.6 combination readily dissolves hydrophobic contami-
Comparative studies have shown that buffered formalin is nants, such as lipids and lipoproteins; collects cell debris
the least damaging among tested fixatives, with mercury- and strips away most DNA-associated proteins (Fig.
based fixatives, such as Bouin’s and B-5, being the 3.1). Isolation of small amounts of DNA from challeng-
worst for DNA recovery (see Table 3.2).7,8 DNA quality ing samples such as fungi can be facilitated by pretreat-
will also depend on how the tissue was handled prior to ment with cetyltrimethylammonium bromide, a cationic
fixation and the length of time the tissue was in fixation. detergent that efficiently separates DNA from polysac-
In general, DNA target products of 100 base pairs (bp) charide contamination. To avoid RNA contamination,
or less can consistently be obtained from fixed tissue. RNase, an enzyme that degrades RNA, can be added at
Extended digestion in proteinase K may yield longer this point. Alternatively, RNase may also be added to the
fragments. resuspended DNA at the end of the procedure.
82 Section II • Common Techniques in Molecular Biology

DNA in DNA
aqueous precipitation
solution (ethanol)
Lysis Acidification Extraction
(NaOH, SDS) (acetic acid, (phenol,
salt) chloroform)
Cells in
suspension Organic
Lysed cells
Cell debris phase

DNA

FIGURE 3.1 General scheme of organic DNA isolation. After lysis of cells, cellular contents are acidified, and particulate matter
is pelleted and extracted with phenol and chloroform. This emulsion will separate into two phases. The upper aqueous phase con-
tains the DNA that can be precipitated by mixing with ethanol. Collecting the precipitated DNA by centrifugation and resuspending
it in a small volume of buffer or water (50 to 250 μL) increases the DNA concentration of the isolated DNA.

A biphasic emulsion forms when phenol and chloro-

form are added to the hydrophilic suspension of lysed ethanol cannot be distilled. The isopropanol used
cells (lysate). At the proper pH, centrifugation will settle is undenatured, or pure, because it is composed
the hydrophobic phase on the bottom, with the hydro- of 99% isopropanol and 1% water, with no other
philic phase on top. Lipids and other hydrophobic com- components.
ponents will dissolve in the lower hydrophobic phase. The choice of which alcohol to use depends on
DNA will dissolve in the upper aqueous phase. Amphi- the starting material, the size and amount of DNA
philic components, which have both hydrophobic and to be isolated, and the design of the method. Iso-
hydrophilic properties and cell debris, will collect as a propanol is less volatile than ethanol and precip-
white precipitate at the interface between the two layers. itates DNA at room temperature. Precipitation at
The DNA in the upper phase is then precipitated by room temperature reduces coprecipitation of salt.
mixing with ethanol or isopropanol and salt (ammo- Also, compared with ethanol, less isopropanol is
nium, potassium or sodium acetate, or lithium or sodium added for precipitation; therefore, isopropanol can
chloride). The ethyl or isopropyl alcohol is added to the be more practical for large-volume samples. For
upper-phase solution at 2:1 or 1:1 ratios, respectively, low concentrations of DNA, longer precipitation
and the DNA forms a solid precipitate. times at freezer temperatures may be required to
maximize the amount of DNA that is recovered.
An important consideration to precipitating the
DNA at freezer temperatures is that the increased
viscosity of the alcohol at low temperatures will
Advanced Concepts require longer centrifugation times to pellet the
DNA.
Both ethanol and isopropanol are used for molec-
ular applications. The ethanol is one of the gen-
eral-use formulas, reagent grade. Reagent-grade
alcohol (90.25% ethanol, 4.75% methanol, 5% Recovery of minimal amounts of DNA can be opti-
isopropanol) is denatured; that is, the ethanol is mized using carrier molecules. In early studies, yeast
mixed with other components because pure 100% RNA or glycogen was used to coprecipitate low con-
centrations of DNA.9 More recently, glycogen or linear
 Chapter 3 • Nucleic Acid Extraction Methods 83

polyacrylamide have been used for this purpose. Gly-

cogen is a biological material derived from mussels Advanced Concepts
(e.g., Mytilus edulis). As a result, some preparations of
glycogen may contain DNA.10 Commercial preparations The presence of the chelating agent, EDTA, pro-
may be treated with DNase to avoid nucleic acid con- tects the DNA from damage by DNases present
tamination. Commercial glycogen may also include a in the environment of DNA preparations intended
color indicator to aid in detecting small pellets. Gener- for long-term storage. EDTA is a component of
ally, 10 to 20 micrograms of carrier is added per mil- TE buffer (10 mM Tris, 1 mM EDTA) and other
liliter of isopropanol mixture before incubation and resuspension buffers. The EDTA will also inhibit
centrifugation. enzyme activity when the DNA is used in various
The DNA precipitate is collected by centrifugation. procedures, such as restriction enzyme digestion or
Excess salt is removed by rinsing the pelleted nucleic PCR. One must be careful not to dilute the DNA
acid in 70% ethanol, centrifuging, and discarding the too far so that large volumes (e.g., more than 10%
supernatant, then dissolving the DNA pellet in rehy- of a reaction volume) of the DNA–EDTA solu-
dration buffer, usually 10 mM Tris, 1 mM EDTA (TE), tion are required for subsequent analysis methods.
or water. When DNA yield is low, as is the case with some
clinical samples, it is better to dissolve it in water.
Inorganic Isolation Methods More of this can be used in analysis procedures
without adding excess amounts of EDTA. Because
Safety concerns in the clinical laboratory make the most of or the entire sample will be used for anal-
use of caustic reagents such as phenol undesirable. ysis, protection in storage is not a concern.
Methods of DNA isolation that do not require phenol
extraction have therefore been developed and are used
in many laboratories. Initially, these methods did not
provide the efficient recovery of clean DNA achieved
with phenol extraction; however, newer methods have Advanced Concepts
proven to produce high-quality DNA preparations in
good yields. Precipitation of the DNA excludes hydrophilic
Inorganic DNA extraction is sometimes called proteins, carbohydrates, and other residual con-
“salting out” (Fig. 3.2). It makes use of low-pH and taminants still present after protein extraction. In
high-salt conditions to selectively precipitate proteins, addition, the concentration of the DNA can be
leaving the DNA in solution. The DNA can then be pre- controlled by adjusting the buffer or water volume
cipitated as described previously using isopropanol, pel- used for resuspension of the pellet.
leted and resuspended in TE buffer or water.

DNA in DNA
aqueous precipitation
solution (isopropanol)
FIGURE 3.2 Inorganic DNA isola-
tion does not include the organic Lysis Protein
extraction step. Proteins are precipi- (Tris, EDTA, precipitation
tated from the cell lysate with high Cells in
SDS) (sodium
salt concentration and low pH. The suspension
acetate)
Lysed cells
supernatant containing the DNA is
then mixed with ethanol or isopropa- Protein
nol to precipitate the DNA to be col-
lected and resuspended in a smaller
volume. DNA
84 Section II • Common Techniques in Molecular Biology

Solid-Phase Isolation
Advanced Concepts
More rapid and comparably effective DNA extraction
can be performed using solid matrices to bind and hold Alkaline lysis can be used to specifically select for
the DNA for washing. Silica-based products were shown plasmid DNA because chromosomal DNA will not
to effectively bind DNA in high-salt conditions.11 Many renature properly upon neutralization and form a
variations on this procedure were developed, including precipitate. The denatured chromosomal DNA and
the use of diatomaceous earth as a source of silica par- protein can be removed by centrifugation before
ticles.12 Modern systems can be purchased with solid the supernatant containing plasmid DNA is applied
matrices in the form of columns or beads. Columns come to the column.
in various sizes, depending on the amount of DNA to be
isolated. Columns used in the clinical laboratory are most
often small “spin columns” that fit inside microcentrifuge
tubes. These columns are commonly used to isolate viral Advanced Concepts
and bacterial DNA from serum, plasma, or cerebrospinal
fluid. They are also used routinely for isolation of cellular Solid matrices conjugated to specific sequences of
DNA in genetics and oncology. Preparation of samples nucleic acid can also be used to select for DNA
for isolation of DNA on solid-phase media starts with containing complementary sequences by hybrid-
cell lysis and release of nucleic acids, similar to organic ization. After removal of noncomplementary
and inorganic procedures (Fig. 3.3). Specific buffers are DNA, the bound complementary DNA can be
used to lyse bacterial, fungal, or animal cells. Buffer eluted by heating the matrix or by breaking the
systems designed for specific applications (e.g., bacterial hydrogen bonds chemically.
cell lysis or human cell lysis) are commercially available.

DNA in
aqueous
solution
DNA adsorption
Lysis Acidification (low pH)
(supplied (supplied
reagents) reagents)
Cells in
suspension
Lysed cells

Cell debris
Wash DNA Elute DNA
(supplied (low salt)
buffer)

DNA

FIGURE 3.3 Isolation of DNA on solid media. Before applying to the silicate column, particulates from the cell debris may be
removed by centrifugation. Solid-phase systems include the proprietary buffers and solutions for adhering the DNA on the column,
washing it, and eluting it in a small volume (10 to 100 μL).
 Chapter 3 • Nucleic Acid Extraction Methods 85

For solid-phase separation, the cell lysate is applied

to a column in high-salt buffer, and the DNA in solu-
tion adsorbs to the solid matrix. Carrier RNA or DNA
is applied with the DNA to enhance recovery, prevent-
ing irreversible binding of the small amount of target
DNA. The carrier must be a nucleic acid of more than
200 nucleotides in length in order to bind to the silica
membrane. Other carriers used to enhance precipitation,
such as glycogen and linear polyacrylamide, cannot be
used in this application. After the immobilized DNA
is washed with buffer, the DNA is eluted in a spe-
cific volume of water, TE, or another low-salt buffer.
The washing solutions and the eluant can be drawn
through the column by gravity, vacuum, or centrifugal
force. DNA absorbed to magnetic beads is washed by FIGURE 3.4 Automated DNA isolation systems use car-
suspension of the beads in buffer and collection of the tridges containing lysis, adsorption, and elution agents and
beads using a magnet applied to the outside of the tube magnetic beads. Bar coding of reagents and sample tubes
while the buffer is aspirated or poured off. The DNA IQ assures accurate sample and reagent lot tracking.
system (Promega) uses a magnetic resin that holds a spe-
cific amount of DNA (100 ng). When the DNA is eluted
in 100 μL, the DNA concentration is known (1 ng/μL) either preclude or prohibit extensive DNA purification.
and ready for analysis. These include screening large numbers of samples by
Solid-phase isolation is the methodology employed simple methods (e.g., electrophoresis with or without
for most robotic DNA isolation systems that use restriction enzyme digestion and some amplification
magnetized glass beads or membranes to bind DNA procedures), isolation of DNA from limited amounts of
(Fig. 3.4). These systems are finding increased use in starting material, and isolation of DNA from challeng-
clinical laboratories for the automated isolation of DNA ing samples such as fixed, paraffin-embedded tissues.
from cultures, blood, tissue, bone marrow, plasma, and In these cases, simple lysis of cellular material in the
other body fluids. DNA isolated from specimens, such sample will yield sufficiently useful DNA for amplifica-
as urine and amniotic fluid, has been reported to work tion procedures.
better in PCR applications, possibly due to the removal Simple screening methods are mostly used for
of the inhibitory substances found in these types of research purposes in which large numbers of samples
specimens by the prefiltering step. In most systems, must be processed. In contrast, analysis of paraffin
a measured amount of sample—for example, 200 to samples is frequently performed in the clinical labo-
1,000 μL of whole blood, 100-μL cell suspensions (106 ratory. Fixed tissue is more conveniently accessed in
to 107 cells), or 10 to 50 mg of tissue, in sample tubes— the laboratory and may sometimes be the only source
is placed into the instrument along with cartridges or of patient material. Thin sections are usually used for
racks of tubes containing the reagents used for isolation. analysis, although sectioning is not necessary with
Reagents are formulated in sets depending on the type very small samples such as needle biopsies. Paraffin-
and amount of starting material. The instrument is then embedded specimens can be dewaxed with xylene or
programmed to lyse the cells and isolate and elute the other agents and then rehydrated before nucleic acid iso-
DNA automatically. lation, the fixed tissue can be used without dewaxing.
For some tests, such as somatic mutation analyses, an
adjacent stained serial section can be examined micro-
Proteolytic Lysis of Fixed Material
scopically to identify areas of the tissue comprising
Although high-quality DNA preparations are tantamount the appropriate percentage of tumor cells. The identi-
to successful procedures, there are circumstances that fiable areas of the tumor can then be isolated directly
86 Section II • Common Techniques in Molecular Biology

Tissue embedded Rapid Extraction Methods

in paraffin Target cells
With the advent of PCR, new and faster methods of
DNA isolation have been developed. The minimal
sample requirements of amplification procedures allow
for the use of material previously not utilizable for anal-
Paraffin ysis. Rapid lysis methods and DNA extraction/storage
cards provide sufficiently clean DNA that can be used
for amplification.
Chelex is a cation-chelating resin that can be used
for simple extraction of DNA from minimal samples.13
A suspension of 10% Chelex resin beads is mixed with
A Microdissection the specimen, and the cells are lysed by boiling. After
Deparaffinize centrifugation of the suspension, DNA in the supernatant
(xylene, ethanol is cooled and may be further extracted with chloroform
wash)
before use in amplification procedures. This method is
most commonly used in forensic applications14,15 but
5–20 micron
may also be useful for purification of DNA from clinical
sections
samples and fixed, paraffin-embedded specimens16 and
samples spotted on storage cards.17,18
Digest
(proteinase K,
Isolation of Mitochondrial DNA
Tris buffer) There are two approaches to the isolation of mitochon-
drial DNA from eukaryotic cells. One method is to
first isolate the mitochondria by centrifugation. After
B cell preparations are homogenized by grinding on ice,
FIGURE 3.5 Crude extraction of DNA from fixed, paraf- the homogenate is centrifuged at low speed (700 to
fin-embedded tissue. Selected regions of tissue are scraped 2,600 × g) to pellet intact cells, nuclei, and cell debris.
from slides (macrodissection; A) and extracted in proteinase K The mitochondria are pelleted from the supernatant in a
(B). second high-speed centrifugation (10,000 to 16,000 × g)
and lysed with detergent. The lysate is treated with pro-
teinase to remove protein contaminants. Mitochondrial
DNA can then be precipitated with cold ethanol and
from the slide by lifting or scraping in buffer with or resuspended in water or appropriate buffers for analysis.
without the use of a microscope (microdissection or The second approach to mitochondrial DNA prepara-
macrodissection, respectively; Fig. 3.5) or laser capture tion is to isolate total DNA as described previously. The
microscopy. preparation will contain mitochondrial DNA that can be
Before lysis, cells may be washed by suspension analyzed within the total DNA background by hybrid-
and centrifugation in saline or other isotonic buffers. ization or PCR.
Reagents used for cell lysis depend on the subsequent
use of the DNA. For simple screens, cells can be lysed
in detergents, such as SDS or Triton. For use in PCR Advanced Concepts
amplification, cells may be lysed in a mixture of Tris
buffer and proteinase K. The proteinase K will digest When homogenizing cells for isolation of mito-
proteins in the sample, lysing the cells and inactivating chondria, care must be taken not to overgrind the
other enzymes. The released DNA can be used directly tissue and dissociate the mitochondrial membranes.
in the amplification reaction.
 Chapter 3 • Nucleic Acid Extraction Methods 87

Specimen Collection
Grinding in alkaline buffers with reducing agents
such as β-mercaptoethanol will protect the mito- RNA tests, especially those that involve gene expres-
chondria during the isolation process. A high-ion- sion, such as array analysis or quantitative reverse
ic-strength buffer can also be used to selectively transcriptase PCR, are most accurate when the RNA
lyse the nuclear membranes. is stabilized from further metabolism or degradation
after collection. Specialized collection tubes have been
devised to stabilize RNA immediately upon blood draw.
In the laboratory, tissue or cell pellets can be stored and/
or transported in preservative reagents.13,19,20

ISOLATION OF RNA Advanced Concepts

Working with RNA in the laboratory requires strict pre-
Several chemical methods have been developed to
cautions to avoid sample RNA degradation. RNA is espe-
inactivate or eliminate RNases. Diethyl pyrocar-
cially labile due to the ubiquitous presence of RNases.
bonate (DEPC) can be added to water and buffers
These enzymes are small proteins that can renature,
(except for Tris buffer) to inactivate RNases per-
even after autoclaving, and regain activity. They remain
manently. DEPC converts primary and second-
active at a wide range of temperatures (down to –20°C
ary amines to carbamic acid esters. It cross-links
and below). Unlike DNases, RNases must be eliminated
RNase proteins through intermolecular covalent
or inactivated before isolation of RNA.
bonds, rendering them insoluble. Because of its
It is necessary to allocate a separate RNase-free
effect on amine groups, DEPC will adversely
(RNF) area of the laboratory for storage of materials and
affect Tris buffers. DEPC will also interact with
specimen handling intended for RNA analysis. Gloves
polystyrene and polycarbonate and is not recom-
must always be worn in the RNF area. Disposables
mended for use on these types of materials.
(tubes, tips, etc.) that come in contact with the RNA
Other RNase inhibitors include vanadyl-
should be kept at this location and never be touched by
ribonucleoside complexes, which bind the active
ungloved hands. Articles designated DNAse-free/RNF
sites of the RNase enzymes, and macaloid clays,
by suppliers may be used directly from the package.
which absorb the RNase proteins. Ribonuclease
Although reusable glassware is seldom used for RNA
inhibitor proteins can be added directly to RNA
work, it can be rendered RNF. After cleaning, glassware
preparations. These proteins form a stable nonco-
must be baked for four to six hours at 400°C to inactivate
valent complex with the RNases in solution. Some
the RNases.
of these interactions require reducing conditions;
therefore, dithiothreitol might be added in addition
Total RNA to the inhibitor.
H2 O O O CH2
There are several types of RNA in prokaryotes and
C C C C
eukaryotes. The most abundant (80% to 90%) RNA in
all cells is ribosomal RNA (rRNA). This RNA consists O O
of two components, large and small, which are visu- Diethyl pyrocarbonate.
alized by agarose gel electrophoresis (see Fig. 3.10).
Depending on the cell type and conditions, the next most
abundant RNA fraction (2.5% to 5%) is messenger RNA
RNA Isolation Chemistries
(mRNA). This mRNA may be detected as a faint back-
ground underlying the rRNA detected by agarose gel Preparation of specimen material for RNA extraction
electrophoresis. Transfer RNA and small nuclear RNAs differs in some respects from DNA extraction. Retic-
are also present in the total RNA sample. ulocytes in blood and bone marrow samples are lysed
88 Section II • Common Techniques in Molecular Biology

by osmosis or separated from WBCs by centrifugation. is added at the lysis step to eliminate contamination of
When dissociating tissue, the sample should be kept DNA. Alternatively, RNF DNase also may be added
frozen in liquid nitrogen or immersed in buffer that will directly to the isolated RNA at the end of the procedure.
inactivate intracellular RNases. This is especially true After phase separation, the upper aqueous phase contain-
for tissues such as pancreas that contain large amounts ing the RNA is removed to a clean tube, and the RNA is
of innate RNases. Bacterial and fungal RNA are also iso- precipitated by addition of two volumes of ethanol or one
lated by chemical lysis or by grinding in liquid nitrogen. volume of isopropanol. Glycogen or yeast-transfer RNA
Viral RNA can be isolated directly from serum, plasma, may be added at this step as a carrier to aid RNA pellet
culture medium, or other cell-free fluids by means of formation. The RNA precipitate is then washed in 70%
specially formulated spin columns or beads. Cell-free ethanol and resuspended in RNF buffer or water.
material is used for these isolations because most total
RNA isolation methods cannot distinguish between Solid-Phase Isolation
RNA from microorganisms and those from host cells.
Solid-phase separation of RNA begins with similar
steps as described previously for organic extraction. The
Organic Isolation
strong denaturing buffer conditions must be adjusted
The cell lysis step for RNA isolation is performed in deter- before application of the lysate to the column (Fig. 3.7).
gent or phenol in the presence of high salt (0.2 to 0.5 M In some procedures, ethanol is added at this point. Some
NaCl) or RNase inhibitors. Guanidine isothiocyanate systems provide a filter column to remove particulate
(GITC) is a strong denaturant of RNases and can be used material before application to the adsorption column.
instead of high-salt buffers. Strong reducing agents such As with DNA columns, commercial reagents are sup-
as 2-mercaptoethanol may also be added during this step. plied with the columns to optimize RNA adsorption and
Once the cells are lysed, proteins can be extracted by washing on the silica-based matrix.
organic means (Fig. 3.6). Acid phenol:chloroform:iso- After lysate is applied to the column in the high-salt
amyl alcohol (25:24:1) solution efficiently extracts RNA. chaotropic buffer, the adsorbed RNA is washed with the
Chloroform enhances the extraction of the nucleic acid supplied buffers. DNase may be added directly to the
by denaturing proteins and promoting phase separation. adsorbed RNA on the column to remove contaminat-
Isoamyl alcohol prevents foaming. For RNA, the organic ing DNA. Washing solutions and the eluant are drawn
phase must be acidic (pH 4 to 6). The acidity of the through the column by gravity, vacuum, or centrifu-
organic phase is adjusted by overlaying it with a buffer of gal force. Small columns of silica-based material that
the appropriate pH. In some isolation procedures, DNase fit inside microfuge tubes (spin columns) are widely

RNA in RNA
aqueous precipitation
solution) (ethanol)
Lysis Extraction
(guanidinium (phenol,
isothiocyanate) chloroform)
Cells in
suspension
Lysed cells Organic
phase

RNA

FIGURE 3.6 Organic extraction of total RNA is similar to that of DNA; however, the RNA must be protected from intracellular
and extracellular RNases. The RNA in solution is precipitated and resuspended, similar to DNA. Some procedures include treat-
ment of the RNA solution with DNase to remove any contaminating DNA.
 Chapter 3 • Nucleic Acid Extraction Methods 89

RNA adsorption
Lysis (low pH)
(supplied
reagents)
Cells in
suspension
Lysed cells

Wash RNA Elute RNA

(supplied
buffer)

RNA

FIGURE 3.7 Isolation of RNA on a solid matrix is also similar to DNA isolation. Buffers and wash solutions are designed for the
RNA target.

used for routine nucleic acid isolation from all types spin columns for removal of genomic DNA contamina-
of specimens. The eluted RNA is usually of sufficient tion are included in some systems. Automated isolation
concentration and purity for direct use in most appli- systems also have reagent kits and cartridges designed to
cations. Automated systems like those shown in Figure isolate RNA from fixed tissue.
3.4 use magnetic beads or particles for RNA isolation as
well. Special reagent sets are available for RNA or total Isolation of polyA (Messenger) RNA
nucleic acid (RNA and DNA) on these instruments.
As previously stated, approximately 80% to 90% of total
Generally, 1 million eukaryotic cells or 10 to 50 mg
RNA is rRNA. Often the RNA required for analysis is
of tissue will yield about 10 μg of RNA. The yield of
mRNA, accounting for only about 2.5% to 5% of the
RNA from biological fluids will depend on the con-
total RNA yield. The majority of mRNA consists of
centration of microorganisms or other target molecules
mRNA from highly expressed genes. Rare or single-copy
present in the specimen (Table 3.3).
mRNA is, therefore, a very minor part of the total RNA
isolation. To enrich the yield of mRNA, especially rare
Proteolytic Lysis of Fixed Material
transcripts, protocols employing single-stranded oligo-
The quality of RNA from fixed, paraffin-embedded mers of thymine or uracil immobilized on a matrix resin
tissue will depend on the fixation process and han- column or beads are used (Fig. 3.8). The polyT or polyU
dling of the specimen before fixation (Table 3.2).21,22 oligomers will bind the polyA tail found exclusively on
Commercial reagent sets are available for extraction of mRNA. After washing away residual RNA, polyA RNA
RNA from fixed-tissue specimens. These reagents are is eluted by washing the column with warmed, low-salt
designed to provide lysis and incubation conditions that buffer containing detergent to break the hydrogen bonds
reverse formaldehyde modification of RNA and effi- between the mRNA and the column. With this approach,
ciently release RNA from tissue sections while avoid- approximately 30 to 40 ng of mRNA can be obtained
ing further RNA degradation. Specialized reagents or from 1 μg of total RNA.
90 Section II • Common Techniques in Molecular Biology

oligomer. Also, mRNAs with short polyA tails may

TABLE 3.3 Yield of RNA From Various not bind efficiently or at all. AT-rich DNA fragments
Specimen Sources36-38 might bind to the column and not only compete with
the desired mRNA target but also contaminate the final
Specimen Expected Yield* eluant. Potential digestion of the oligo-conjugated matri-
Blood† (1 mL, 3.5–10  × 106 WBCs/mL) 1–10  μg ces precludes the use of DNase on the RNA before it is
bound to the column. Treatment of the eluant with RNF
†
Buffy coat (1 mL whole blood) 5–10  μg DNase is possible, but the DNase should be inactivated
Bone marrow† (1 mL) 50–200  μg
if the mRNA is to be used in procedures involving DNA
components. Furthermore, rRNA may co-purify with
Cultured cells‡ (107 cells) 50–150  μg the polyA RNA. The final product, then, is enriched in
polyA RNA but is not a pure polyA preparation.
Buccal cells (1 mg) 1–10  μg

Solid tissue§ (1 mg) 0.5–4  μg

MEASUREMENT OF NUCLEIC ACID QUALITY
||
Fixed tissue (1 mm ) 3
0.2–3  μg
AND QUANTITY
Bacterial culture¶ (0.5 mL, 0.7 10–100  μg
absorbance units) Laboratory analysis of nucleic acids produces vari-
able results, depending on the quality and quantity of
*Specimen handling especially affects RNA yield. Isolation of polyA RNA will
result in much lower yields. See text.
input material. This is an important consideration in the
†
RNA yield will depend on WBC count. medical laboratory because test results must be accu-
‡

§
RNA yield will depend on type of cells and the conditions of cell culture. rately interpreted with respect to disease pathology.
Liver, spleen, and heart tissues yield more RNA than brain, lung, ovary,
kidney, or thymus tissues.
Consistent results require that run-to-run variation be
||
Isolation of RNA from fixed tissue is especially affected by the type of fixative minimized. Fortunately, measurement of the quality and
used and the age and the preliminary handling of the original specimen.
¶
quantity of DNA and RNA is straightforward.
Different bacterial types and fungi will yield more or less RNA.

Electrophoresis
DNA and RNA can be analyzed for quality by resolv-
ing an aliquot of the isolated sample on an agarose gel
mRNA (Fig. 3.9). Fluorescent dyes such as ethidium bromide or
5′ A A A A A A A A A 3′ SybrGreen I bind specifically to DNA and are used to
3′ T T T T T T T T T 5′
visualize the sample preparation. Ethidium bromide or
SybrGreen II are used to detect RNA. Less frequently,
silver stain has been used to detect small amounts of
Bead or column DNA by visual inspection.
FIGURE 3.8 Oligo polythymine (or polyuracil) columns or The appearance of DNA on agarose gels depends
beads bind the polyA tail of mRNA. Peptide nucleic acid dT or on the type of DNA isolated. A good preparation of
dU can also be used. These polymers are less subject to degra- plasmid DNA will yield a bright signal from supercoiled
dation by contaminating nucleases. plasmid DNA with minor or no other bands that rep-
resent nicked or broken plasmid (see Fig. 3.10). High-
molecular-weight chromosomal DNA should collect as a
There are limitations to the isolation of polyA RNA bright band near the top of the gel. A high-quality prepa-
using oligo dT or dU. The efficiency of polyA and ration of RNA will yield two distinct bands of ribosomal
polyU binding is variable. Secondary structure (intra- RNA. The integrity of these bands is an indication of the
strand or interstrand hydrogen bonds) in the target integrity of the other RNA species present in the same
sample may compete with binding to the capture sample. If these bands are degraded (smeared) or absent,
 Chapter 3 • Nucleic Acid Extraction Methods 91

L N, SC

M SC N L Nicked/relaxed

23 kb

Linear
0.6 kb Supercoiled

FIGURE 3.9 After agarose gel electrophoresis, compact supercoiled plasmid DNA (SC) will travel farther through the gel than
nicked plasmid (N), which has single-strand breaks. Relaxed plasmid DNA (R) has double-strand breaks and will migrate accord-
ing to its size, 23 kb in the drawing on the left. Linear (L) plasmids migrate according to the size of the plasmid. A gel photo shows
a plasmid preparation. N, nicked; SC, supercoiled; L, linear; R, relaxed; M, molecular-weight markers.

Wells

Genomic
DNA
28S rRNA

18S rRNA

FIGURE 3.10 Intact ethidium bromide–stained human chromosomal DNA (left) and total RNA (right) after agarose gel electro-
phoresis. High-quality genomic DNA runs as a tight smear close to the loading wells. High-quality total RNA appears as two rRNA
bands representing large and small ribosomal RNA species (shown with molecular weight markers, M).

the quality of the RNA in the sample is deemed unac- with that of a known amount of control DNA or RNA
ceptable for use in molecular assays. loaded on the same gel. Densitometry of the band inten-
When fluorescent dyes are used, DNA and, less accu- sities gives the most accurate measurement of quantity.
rately, RNA can be quantified by comparison of the flu- For some procedures, estimation of DNA or RNA quan-
orescence intensity of the sample aliquot run on the gel tity can be made by visual inspection.
92 Section II • Common Techniques in Molecular Biology

Spectrophotometry
EXAMPLE 2: An RNA preparation diluted 1/10
Nucleic acids absorb light at 260 nm through the adenine
yields an absorbance reading of 0.500 at 260 nm.
residues. Using the Beer–Lambert law, concentration
The concentration is
can be determined from the absorptivity constants
(50 for double-stranded DNA, 40 for RNA). The rela- 0.500 absorbance units × 40 μg mL
tionship of concentration to absorbance is expressed as per absorbance unit × 10 = 200 μg mL
A = ∈bc The yield of the sample is calculated using the
where A = absorbance, ∈ = molar absorptivity (L/mol- volume of the preparation. If the DNA was eluted
cm), b = path length (cm), and c = concentration (mg/L). or resuspended in 0.2 mL in the case illustrated
The absorbance at this wavelength is thus directly previously, the yield would be
proportional to the concentration of the nucleic acid 200 μg mL × 0.2 mL = 40 μg
in the sample. Using the absorptivity as a conversion
factor from optical density to concentration, one optical
density unit (or absorbance unit) at 260 nm is equivalent
to 50 mg/L (or 50 μg/mL) of double-stranded DNA and
40 μg/mL of RNA. To determine concentration, multiply Histooricaal Higghlligghtts
the spectrophotometer reading in absorbance units by
the appropriate conversion factor. Phenol absorbs ultra- The maximum absorption of light at 260 nm was
violet light at 270 to 275 nm, close to the wavelength of one of the clues suggesting that DNA was the
maximum absorption by nucleic acids. This means that molecular matter of genetic material. In 1942,
residual phenol from organic isolation procedures can Lewis Stadler reported results on studies of the
increase 260 readings, so phenol contamination must be effect of wavelength of ultraviolet (UV) light on
avoided when measuring concentration at 260 nm. mutagenesis in corn plants.23 He observed that
If the DNA or RNA preparation is of sufficient con- the most mutagenic monochromatic light had a
centration to require dilution before spectrophotometry wavelength of 260 nm. Together with the data of
in order for the reading to fall within the linear reading Avery24 and observations of Hershey and Chase,25
range, the dilution factor must be included in the calcu- Stadler ’s observations further supported the idea
lation of quantity. Multiply the absorbance reading by that DNA is the carrier of genetic information.
the conversion factor and the dilution factor to find the
concentration of nucleic acid.

Spectrophotometric measurements may also be used to

EXAMPLE 1: A DNA preparation diluted /100 1 estimate the purity of nucleic acid. Ideally, contamina-
yields an absorbance reading of 0.200 at 260 nm. tion can be detected by reading the concentration over a
To obtain the concentration in μg/mL, multiply: range of wavelengths. Graphing the absorbance reading
as a function of wavelength produces a curve or spec-
0.200 absorbance units × 50 μg mL trum that should peak at A260 nm for nucleic acid. Some
per absorbance unit × 100 = 1, 000 μgg mL spectrophotometers produce this graph automatically.
The yield of the sample is calculated using the Even with such automation, however, spectral analy-
volume of the preparation. If the DNA was eluted sis for each specimen sample is not practical for most
or resuspended in a volume of 0.5 mL in the case routine medical laboratory work. In practice, nucleic
illustrated previously, the yield would be acid solutions are read at two or three distinct wave-
lengths (Table 3.4). Absorbance over background at any
1, 000 μg mL × 0.5 mL = 500 μg wavelength other than the A260 maxima of the nucleic
acid indicates contamination.
 Chapter 3 • Nucleic Acid Extraction Methods 93

fragmented or degraded DNA will produce an A260

TABLE 3.4 Common Contaminants and Their reading comparable to intact DNA. In this regard, fluo-
Wavelengths of Peak Absorbance rometry is considered by some to be a better method of
quality assessment.
Wavelength (nm) Contaminant

230 Organic compounds Histooricaal Higghlligghtts

270 Phenol
Estimation of protein contamination in nucleic
280 Protein acid is based on a method by Warburg and Chris-
tian designed to measure nucleic acid contamina-
>330 Particulate matter tion in protein.26 This method utilizes the natural
light absorption at 280 nm of the aromatic amino
acid side chains, tryptophan, and tyrosine. Nucleic
Routine nucleic acid isolation procedures produce acids are common contaminants of protein prepa-
reasonably pure DNA solutions free from particles, rations and naturally absorb light at 260 nm,
organic compounds, and phenol. Due to its abundance where there is no interference by protein. In the
and close association with nucleic acid in the cell, the Warburg–Christian method, the test material was
most likely contaminant in a nucleic acid preparation read at 260 and 280 nm, and then a nomograph
will be protein. Protein absorbs light at 280 nm through was used to determine the relative amounts of
the aromatic tryptophan and tyrosine residues. Although protein and nucleic acid.
this measurement is not highly accurate, general ranges
indicate at least the presence of protein contaminants.
The absorbance of the nucleic acid at 260 nm should be
1.6 to 2.00 times more than the absorbance at 280 nm. Advanced Concepts
If the 260-nm/280-nm ratio is less than 1.6, the nucleic
acid preparation may be contaminated with unaccept- Ultraviolet spectrophotometers dedicated to
able amounts of protein and not of sufficient purity for nucleic acid analysis can be programmed to cal-
use. Such a sample can be improved by column purifi- culate absorbance ratios and concentrations. The
cation, reprecipitating the nucleic acid, or repeating the operator must enter the type of nucleic acid, the
protein-removal step of the isolation procedure. It should dilution factor, and the desired conversion factor.
be noted that low pH affects the 260-nm/280-nm ratio. The instrument will automatically read the sample
Somewhat alkaline buffers (pH 7.5) are recommended at the appropriate wavelengths and do the required
for accurate determination of purity. RNA affords a calculations, giving a reading of concentration in
somewhat higher 260-nm/280-nm ratio, 2.0 to 2.3. A μg/mL and a 260-nm/280-nm ratio.
DNA preparation with a ratio higher than 2.0 may be
contaminated with RNA. Some procedures for DNA
analysis are not affected by contaminating RNA, in
Fluorometry
which case the DNA is still suitable for use. If, however,
RNA may interfere or react with DNA-detection compo- Fluorometry, or fluorescent spectroscopy, measures flu-
nents, RNase should be used to remove the contaminat- orescence related to DNA concentration in association
ing RNA. Because it is difficult to detect contaminating with DNA-specific fluorescent dyes. Early methods
DNA in RNA preparations, RNA should be treated with used 3,5-diaminobenzoic acid 2HCl (DABA).27 This
RNF DNase where DNA contamination may interfere dye combines with alpha-methylene aldehydes (deoxy-
with subsequent applications. ribose) to yield a fluorescent product. It is still used for
Absorbance ratios do not necessarily predict success- the detection of nuclei and as a control for hybridiza-
ful amplification or hybridization reactions. Because tion and spot integrity in microarray analyses. Although
the light is absorbed by single-nitrogen-base moieties, less convenient to use, fluorometry is recommended for
94 Section II • Common Techniques in Molecular Biology

procedures where accurate measurement of intact DNA Absorption and fluorometry readings may not always
is critical, such as next-generation sequencing. agree. First, the two detection methods recognize differ-
More modern procedures use the DNA-specific dye ent targets. Single nucleotides do not bind to fluorescent
Hoechst 33258. This dye combines with adenine-thymine dyes, but they can absorb ultraviolet light and affect
base pairs in the minor groove of the DNA double helix spectrometric readings. Furthermore, absorption mea-
and is thus specific for intact double-stranded DNA. The surements do not distinguish between DNA and RNA,
binding specificity for A-T residues, however, complicates so contaminating RNA may be factored into the DNA
measurements of DNA that have unusually high or low GC measurement. RNA does not enhance the fluorescence
content. A standard measurement is required to determine of the fluorescent dyes and is thus invisible to fluoro-
concentration by fluorometry, and this standard must have metric detection. In fact, specific detection of RNA in
GC content similar to that of the samples being measured. the presence of DNA in solution is not yet possible.
Calf thymus DNA (GC content = 50%) is often used as a Deciding which instrument to use is at the discretion
standard for specimens with unknown DNA GC content. of the laboratory. Most laboratories use spectrophotom-
Fluorometric determination of DNA concentration using etry because the samples can be read directly without
Hoechst dye is very sensitive (down to 200 ng DNA/mL). staining or mixing with dye. For methods that require
PicoGreen and OliGreen are other DNA-specific dyes accurate measurements of low amounts of DNA or RNA
that can be used for fluorometric quantification. Due to (in the range of 10 to 100 ng/mL), fluorometry may
brighter fluorescence upon binding to double-stranded be preferred.
DNA, PicoGreen is more sensitive than Hoechst dye
(detection down to 25 pg/mL concentrations). Like
Microfluidics
Hoechst dye, single-stranded DNA and RNA do not
bind to PicoGreen. OliGreen is designed to bind to short Lab-on-a-chip technology has been applied to nucleic
pieces of single-stranded DNA (oligonucleotides). This acid quantification and analysis using microfluidics-
dye will detect down to 100 pg/mL of single-stranded based instruments. To make a measurement, the sample
DNA. OliGreen will not fluoresce when bound to is applied to a multiwell chip. The sample then moves
double-stranded DNA or RNA. through microchannels across a detector. The instru-
RNA may be measured in solution using SybrGreen ment software generates images in electropherogram
II RNA gel stain.28 The intensity of SyBrGreen II flu- (peak) or gel (band) configurations. For RNA, a quan-
orescence is 20% to 26% lower with polyadenylated tification estimate called the RNA integrity number is
RNA than with total RNA. The sensitivity of this dye determined as a standard measure of RNA integrity. The
is 2 ng/mL. SybrGreen II, however, is not specific to presence of ribosomal impurities in mRNA preparations
RNA and will bind and fluoresce with double-stranded may also be calculated. This system is more automated
DNA as well. Contaminating DNA must therefore be than standard spectrophotometry or fluorometry, uses a
removed in order to get an accurate determination of minimal volume of sample (as low as 1 μL), and can
RNA concentration. test multiple samples simultaneously. Microfluidics
Fluorometry measurements require calibration of the is useful for the analysis of studies on small RNAs,
instrument with a known amount of standard before mea- such as microRNAs in eukaryotes and gene expression
surement of the sample. For methods using Hoechst dye, in bacteria.
the dye, diluted to a working concentration of 1 μg/mL
in water, is mixed with the sample (usually a dilution
of the sample). Once the dye and sample solution are
mixed, fluorescence must be read within two hours STUDY QUESTIONS
because the dye/sample complex is stable only for
approximately this amount of time. The fluorescence is
DNA Quantity/Quality
read in a quartz cuvette. A programmed fluorometer will
read out a concentration based on the known standard 1. Calculate the DNA concentration in μg/mL from the
calibration. following information:
 Chapter 3 • Nucleic Acid Extraction Methods 95

a. Absorbance reading at 260 nm from a 2. If the volume of the RNA solutions in question 1
1:100 dilution = 0.307 was 0.5 mL, calculate the yield for (a)–(d).
b. Absorbance reading at 260 nm from a
1:50 dilution = 0.307 3. An RNA preparation has the following absorbance
c. Absorbance reading at 260 nm from a readings:
1:100 dilution = 0.172
A260 = 0.208
d. Absorbance reading at 260 nm from a
A280 = 0.096
1:100 dilution = 0.088
Is this RNA preparation satisfactory for use?
2. If the volume of the DNA solutions in question 1
was 0.5 mL, calculate the yield for (a)–(d).
4. A blood sample was held at room temperature for
5 days before being processed for RNA isolation.
3. Three DNA preparations have the following A260 and
Will this sample likely yield optimal RNA?
A280 readings:
5. Name three factors that will affect the yield of RNA
Sample OD260 OD280 from a paraffin-embedded tissue sample.
(i) 1 0.419 0.230
(ii) 2 0.258 0.225
References
(iii) 3 0.398 0.174
1. Mirsky A. The discovery of DNA. Scientific American 1968;218:
For each sample, based on the A260/A280 ratio, is each 78–88.
preparation suitable for further use? If not, what is 2. Meselson M, Stahl FW. The replication of DNA in Escherichia
coli. Proceedings of the National Academy of Sciences 1958;44:
contaminating the DNA? 671–682.
3. Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating liquid
4. After agarose gel electrophoresis, a 0.5-microgram biopsies into the management of cancer. Nature Reviews: Clinical
aliquot of DNA isolated from a bacterial culture Oncology 2017;14(9):531–548.
produced only a faint smear at the bottom of the gel 4. Grskovic M, Hiller DJ, Eubank LA, Sninsky JJ, Christopherson C,
Collins JP, Thompson K, Song M, Wang YS, Ross D, Nelles MJ,
lane. Is this an acceptable DNA sample? Yee JP, Wilber JC, Crespo-Leiro MG, Scott SL, Woodward RN.
Validation of a clinical-grade assay to measure donor-derived cell-
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concentration by spectrophotometry with analysis by lar Diagnostics 2016;18:890–902.
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1. Calculate the RNA concentration in μg/mL from the 6. Lucena-Aguilar G, Sánchez-López AM, Barberán-Aceituno C,
following information: Carrillo-Ávila JA, López-Guerrero JA, Aguilar-Quesada R. DNA
source selection for downstream applications based on DNA quality
a. Absorbance reading at 260 nm from a indicators analysis. Biopreservation and Biobanking 2016;14:
1:100 dilution = 0.307 264–270.
b. Absorbance reading at 260 nm from a 7. Greer C, Peterson SL, Kivat NB, Manos MM. PCR amplification
1:50 dilution = 0.307 from paraffin-embedded tissues. American Journal of Clinical
c. Absorbance reading at 260 nm from a Pathology 1991;95:117–124.
8. Perry C, Chung JY, Ylaya K, Choi CH, Simpson A, Matsumoto
1:100 dilution = 0.172 KT, Smith WA, Hewitt SM. A buffered alcohol-based fixative for
d. Absorbance reading at 260 nm from a histomorphologic and molecular applications. Journal of Histo-
1:100 dilution = 0.088 chemistry and Cytochemistry 2016;64:425–440.