Basic DNA and RNA Protocols

493 Pages·1996·30.71 MB·English

by  Adrian J. Harwood

CHAPTER 1
The Simultaneous Isolation
of RNA and DNA
from Tissues and Cultured Cells
Frank Merante, Sandeep Raha,
Juta K. Reed, and Gerald Proteau
1. Introduction
Many techniques are currently available that allow the isolation of
DNA (I-7) or RNA (8-231, but such methods allow only the purification
of one type of nucleic acid at the expense of the other. Frequently, when
cellular material is limiting, it is desirable to isolate both RNA and DNA
from the same source. Such is the case for biopsy specimens, primary
cell lines, or manipulated embryonic stem cells.
Although several procedures have been published that address the need
to simultaneously purify both RNA and DNA from the same source
(2&31), most methods are simply a modification of the original proce-
dure of Chirgwin et al. (8). Such procedures utilize strong chaotropic
agents, such as guanidinium thiocyanate and cesium trifluoroacetate
(25,2 7), to simultaneously disrupt cellular membranes and inactivate
potent intracellular RNases (26,29,30). The limitations of such tech-
niques are the need for ultracentrifugation (26-28,30) and long process-
ing times (ranging 1644 h).
Methods for isolating both RNA and DNA that circumvent the
ultracentrifugation step take advantage of the fact that phenol (1,32) can
act as an efficient deproteinization agent quickly disrupting cellular
integrity and denaturing proteins (24,31). The method presented here
From Methods m Molecular Brology, Vol 58 Basic DNA and RNA Protocols
Edlted by A Harwood Humana Press Inc , Totowa, NJ
3
4 Merante et al.
takes advantage of the qualities offered by phenol extraction when it is
coupled with a suitable extraction buffer and a means for selectively sepa-
rating high-mol-wt DNA from RNA (31).
The method utilizes an initial phenol extraction coupled with two
pheno1:chlorofor-m extractions to simultaneously remove proteins and
lipids from nucleic acid containing solutions. In addition, the constitu-
ents of the aqueous extraction buffer are optimized to increase nucleic
acid recovery, as discussed by Wallace (33). For example, the pH of the
buffer (pH 7.9, the presence of detergent (0.2% SDS), and relatively
low salt concentration (100 mA4 LiCl) allow the efficient partitioning
of nucleic acids into the aqueous phase and the dissociation of proteins.
In addition, the presence of 10 mM EDTA discourages the formation of
protein aggregates (33) and chelates Mg 2+, thereby inhibiting the action
of magnesium dependent nucleases (34).
This method differs from that presented by Krieg et al. (24) in that the
lysis and extraction procedurei s gentle enought o allow the selectiver emoval
of high-mol-wt DNA by spooling onto a hooked glass rod (2,34,35) follow-
ing ethanol precipitation. This avoids additional LiCl precipitation steps
following the recovery of total nucleic acids. Finally, the procedure can be
scaled up or down to accommodate various sample sizes, hence allowing
the processingo f multiple samplesa t onet ime. The approximate time required
for the isolation of total cellular RNA and DNA is 2 h. Using this method
nucleic acids have been isolated from PC12 cells and analyzed by South-
em and Northern blotting techniques (31).
2. Materials
Molecular biology grade reagents should be utilized whenever pos-
sible. Manipulations were performed in disposable, sterile polypropy-
lene tubes whenever possible, otherwise glassware that had been
previously baked at 280°C for at least 3 h was used.
2.1. Nucleic Extraction
from Nonadherent Tissue Culture Cells
1. PBS: 0.137MNaC1,2.68 mMKCl,7.98 mA4Na2HP04, 1.47 mMKH2P04,
pH 7.2.
2. DEPC-treated water: Diethylpyrocarbonate (DEPC)-treated water is pre-
pared by adding 1 mL DEPC to 1 L of double-distilled water (0.1% DEPC
v/v) and stirring overnight. The DEPC is inactlvated by autoclaving at 20
psi for 20 mm (see Note 1).
Isolation of RNA and DNA 5
3. STEL buffer: 0.2% SDS, 10 mMTris-HCl, pH 7.5,lO mMEDTA, and 100
mM LiCl. The buffer is prepared in DEPC-treated water by adding the
Tris-HCl, EDTA, and LiCl components first, autoclaving, andt hen adding
an appropriate volume of 10% SDS. The 10% SDS stock solution 1sp re-
pared by dissolving 10 g SDS in DEPC-treated water and Incubating at
65OC for 2 h prior to use.
4. Phenol: Phenol is equilibrated as described previously (34). Ultrapure,
redistilled phenol, contaimng 0.1% hydroxyquinoline (as an antioxidant),
is initially extracted with 0.5M Tris-HCl, pH 8.0, and then repeatedly
extracted with 0. 1M Tns-HCl, pH 8.0, until the pH of the aqueous phase is
8.0. Then equilibrate with STEL extraction buffer twice prior to use. This
can be stored at 4°C for at least 2 mo.
5. Phenol:chloroform mixture: A 1: 1 mixture was made by adding an equal
volume of chloroform to STEL-equilibrated phenol. Can be stored at 4°C
for at least 2 mo. Phenol should be handled with gloves in a fume hood.
6. 5A4 LiCl: Prepare in DEPC-treated water and autoclave.
7. TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. Prepare in DEPC-
treated water and autoclave.
8. RNA guard, such as RNasin (Promega; Madison, WI).
2.2. Variations for Adherent Cell Cultures and Tissues
9. Trypsm: A 0.125% solution in PBS. For short term store at 4°C; for long
term freeze.
3. Methods
3.1. Nucleic Extraction
from Nonadherent Tissue Culture Cells
In this section we detail nucleic extraction from nonadherent tissue
culture cells. Section 3.2. describes variations of this protocol for adher-
ent cell cultures and tissue.
To prevent RNase contamination from skin, disposable gloves should
be worn throughout the RNA isolation procedure. In addition, it is advis-
able to set aside equipment solely for RNA analysis; for example, glass-
ware, pipets, and an electrophoresis apparatus.
1. Cultured cells (1 x 107) should be cooled on ice (see Note 2). Transfer to
15-mL polypropylene tubes and pellet by centrifugation at 1O Ogf or 5 min.
Wash the cells once with 10 mL of ice-cold PBS and repellet. The pelleted
cells may be left on ice to allow processmg of other samples.
2. Simultaneously add 5 mL of STEL-equilibrated phenol and 5 mL of ice-
cold STEL buffer to the pelleted cells, Gently mix the solution by mver-
6 Merante et al.
slon for 3-5 min, ensuring the cellular pellet is thoroughly dissolved (see
Notes 3 and 4).
3. Centrifuge the mixture at 10,OOOgfo r 5 min at 20°C to separate the phases.
Transfer the aqueous (upper) phase to a new tube using a sterile polypropy-
lene pipet and re-extract twice with an equal volume of phenol:chloroform
(see Note 5).
4. Transfer the aqueous phase to a 50-mL Falcon tube. Differentially precipi-
tate high-mol-wt DNA from the RNA component by addition of 0.1 vol of
ice-cold 5M LiCl and 2 vol of ice-cold absolute ethanol. The DNA will
precipitate immediately as a threaded mass.
5. Gently compact the mass by mixing and remove the DNA by spoolmg
onto a hooked glass rod Remove excess ethanol from the DNA by touch-
mg onto the side of the tube. Remove excess salts by rmsmg the DNA with
1 mL of ice-cold 70% ethanol while still coiled on the rod. Excess ethanol
can be removed by carefully washing the DNA with 1 mL of ice-cold TE,
pH 8.0 (see Note 6).
6. The DNA is then resolublhzed by transferrmg the glass rod mto an appro-
priate volume of TE, pH 8.0 and storing at 4OC.
7. The RNA IS precipitated by placing the tube with the remaimng solution at
-7O”C, or m an ethanol/dry ice bath for 30 mm.
8. Collect the RNA by centritiging at 10,OOOgfo r 15 min. Gently aspirate the
supernatant and rmse the pellet with ice-cold 70% ethanol. Recentrlfuge
for 5 min and remove the supernatant. Dry the RNA pellet under vacuum
and resuspend m DEPC-treated water.
9. For storage as aqueous samples add 5-10 U of RNasm (RNase inhlb-
itor) according to manufacturer’s instructions. Alternatively, the
RNA can be safely stored as an ethanol/LiCl suspension (see Notes 7
and 8).
3.2. Variations for Adherent Cell Cultures and Tissues
3.2.1. Adherent Cells
1. Remove the culture medium from the equivalent of 1 x 1O 7c ells by aspira-
tion and wash the cells once with 10 mL of PBS at 37°C.
2. Add 1 mL of trypsm solution and incubate plates at 37°C until the cells
have been dislodged. This should take approx 10 mm. Dilute the trypsm
solution by addition of ice-cold PBS.
3. Follow Section 3.1.) steps 2-9.
3.2.2. Procedure for Tissue
1, Rinse approx 500 mg of tissue free of blood with ice-cold PBS. Cool and
mince into 3-5-cm cubes with a sterile blade.
Isolation of RNA and DNA
2. Gradually add the tissue to a mortar containing hquid nitrogen and ground
to a tine powder.
3. Slowly add the powdered tissue to an evenly dispersed mixture of 5 mL of
phenol and 5 mL of STEL. This is best accomplished by gradually stirring
the powdered tissue into the phenol:STEL emulsion with a baked glass
rod. Mix the tissue until the components are thoroughly dispersed. Con-
tinue mixmg by gentle inversion for 5 min.
4. Follow Section 3.1.) steps 3-9.
4. Notes
1. DEPC is a suspected carcinogen and should be handled with gloves in a
fume hood. Because it acts by acylating hrstidine and tyrosme residues on
proteins, susceptible reagents, such as Tris solutions, should not be directly
treated with DEPC. Sensitive reagents should simply be made up in DEPC-
treated water as outlined.
2. The integrity of the nucleic acids will be improved by maintaining har-
vested cells or tissues cold.
3. The successo f this procedure hinges on the ability to gently disrupt cellu-
lar integrity while maintaining DNA in an intact, high-mol-wt form. Thus,
mixing of the STEL:phenol should be performed by gentle inversion,
which minimizes shearing forces on the DNA.
4. The proteinaceous interface that partitions between the aqueous
(upper) and phenol phase following the nntial phenol extraction (Sec-
tion 3.1.) step 3) can be re-extracted with phenol:chloroform to improve
DNA recovery.
5. Chloroform is commonly prepared as a 24:l (v/v) mixture with isoamyl
alcohol, which acts as a defoaming agent. We have found that foaming is
not a problem if extractions are performed by gentle inversion or on a
rotating wheel and routinely omit isoamyl alcohol from the mixture.
6. The DNA may be air dried, but will then take longer to resuspend.
7. Followmg the selective removal of high-mol-wt DNA, the remaining RNA
is sufficiently free of DNA contamination such that DNA is not detected
by ethidmm bromide stammg (31). If the purified RNA is to be used for
PCR procedures it 1s strongly recommended that a RNase-treated control
be performed to ensure the absence of contaminatmg DNA. This recom-
mendatron extends to virtually any RNA purification procedure, particu-
larly those involvmg an initial step in which the DNA is sheared.
8. Typical yields of total cellular RNA range between 60-170 ,ug when using
approx 1.5-2 x 1O 7c ells with Az6,jA2s0v alues of approx 1.86 (31). These
values compare favorably with those obtained using guanidinium thiocy-
anate CsCl centnfugatron methods (8).
8 Merante et al.
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11 Chomczynski, P and Sacchi, N (1987) Single-step method of RNA extraction by
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tissues. Anal. Blochem. 174,658-661.
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small numbers of cells. Anal Blochem 173,93-95.
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17. Wilkinson, M (1988) A rapid and convenient method for isolation of nuclear, cyto-
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amplification of RNA by the polymerase chain reaction. Nuclerc Acids Res 17,2 141
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20. Nemeth, G. G., Heydemann, A., and Bolander, M. E. (1989) Isolation and
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21. Verwoerd, T. C., Dekker, B M. M., and Hoekema, A. (1989) A small-scale proce-
dure for the rapid isolation of plant RNAs Nuclezc Acrds Res 17,2362
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23 Tavangar, K , Hoffman, A R., and Kraemer, F. B (1990) A micromethod for the
isolation of total RNA from adipose tissue Anal Bzochem. 186,60-63
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3 1 Raha, S., Merante, F , Proteau, G , and Reed, J. K (1990) Simultaneous tsolation
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Brology Elsevter, New York.

CHAPTER2
Restriction Endonuclease Digestion
of DNA
Duncan R. Smith
1. Introduction
The ability to cleave DNA at specific sites is one of the cornerstones
of today’s methods of DNA manipulation. Restriction endonucleases are
bacterial enzymes that cleave duplex DNA at specific target sequences
with the production of defined fragments. These enzymes can be pur-
chased from the many manufacturers of biotechnology products. The
nomenclature of enzymes is based on a simple system, proposed by Smith
and Nathans (I). The name of the enzyme (such as BarnHI, ,%&I, and
so on) tells us about the origin of the enzyme, but does not give us any
information about the specificity of cleavage (see Note 1). This has to be
determined for each individual enzyme. The recognition site for most of
the commonly used enzymes is a short palindromic sequence, usually
either 4, 5, or 6 bp in length, such as AGCT (for A&I), GAATTC (for
EC&I), and so on. Each enzyme cuts the palindrome at a particular site,
and two different enzymes may have the same recognition sequence, but
cleave the DNA at different points within that sequence. The cleavage
sites fall into three different categories, either flush (or blunt) in which
the recognition site is cut in the middle, or with either 5’- or 3’-over-
hangs, in which case unpaired basesw ill be produced on both ends of the
fragment. For a comprehensive review of restriction endonucleases, see
Fuchs and Blakesley (2).
From Methods fn Molecular Biology, Vol 58 i3a.m DNA and RNA Protocols
Edlted by A Harwood Humana Press Inc , Totowa, NJ
11
12 Smith
2. Materials
1. A 1 OX stock of the appropriate restriction enzyme buffer (see Note 2).
2. DNA to be digested (see Notes 3 and 4) in either water or TE (10 mM Tris-
HCl, pH 8.3, 1 rnMEDTA).
3. Bovine serum albumin (BSA) at a concentration of 1 mg/ mL (see Note 5).
4. Sterile distilled water (see Note 6).
5. The correct enzyme for the digest (see Note 7).
6. 5X loading buffer: 50% (v/v) glycerol, 100 mM Na2EDTA, pH 8,0.125%
(w/v) bromophenol blue (6pb), 0.125% (w/v) xylene cyanol.
7. 100 mM Sperrmdme (see Note 8).
3. Methods
1. Thaw all solutions, with the exception of the enzyme, and then place on ice.
2. Decide on a final volume for the digest, usually between 10 and 50 PL (see
Note 9), and then into a sterile Eppendorf tube, add l/10 vol of reaction
buffer, l/10 vol of BSA, between 0.5 and 1 pg of the DNA to be digested
(see Note 3), and sterile distilled water to the final volume.
3. Take the restriction enzyme stock directly from the -2OOC freezer, and
remove the desired units of enzyme (see Notes 7 and 10) with a clean
sterile pipet tip. Immediately add the enzyme to the reaction and mix (see
Note 11).
4. Incubate the tube at the correct temperature (see Note 12) for approx 1 h.
Genomic DNA can be digested overnight.
5. An aliquot of the reaction (usually l-2 pL) may be mixed with a 5X
concentrated loading buffer and analyzed by gel electrophorests (see
Chapter 3).
4. Notes
1. Enzymes are named according to the system proposed by Smith and
Nathans (1) m which enzymes are named according to the bacteria from
which they are first purified. Therefore, for example, a restriction enzyme
purified from Providencia stuartii, would be identified by the first letter of
the genus name (m this case Provzdencia and hence P) and the first two
letters of the specific epithet (m this case stuartiz and hence st) joined
together to form a three-letter abbreviation-Pst. The first restriction
enzyme isolated from this source of bacteria would therefore be called
PstI (with the number m Roman numerals), and the second P&II, and so
on. Note, however, that the name of the enzyme gives no mformation about
the speciflctty of cleavage, which must be determined from one of the
numerous lists of enzymes and cleavage specificities (the catalog of most
suppliers of restriction enzymes will provide extensive information about