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Rapid isolation of RNA using proteinase K and sodium perchlorate
ANALYTICAL
BIOC HtMlSl
RY 98.
1 Ih-
I??
( 1979)
Rapid Isolation of RNA Using Proteinase K and Sodium Pet-chlorate PAUL M. LIZARDI The
Rdefdkr
AND ALAN ENGELBERG
lJt~i~wsit,v.
Received
Nm.
February
k;w~.
Nru.
l’ori
10021
28, 1979
A simple. efficient procedure for the isolation of cellular nucleic acids is described. It combines the use of sodium dodecyl sulfate. proteinase K, sodium perchlorate, and isopropanol precipitation. The yields and purity of RNA extracted from a variety of sources are comparable or superior to those obtained by phenol extraction. High molecular weight RNA (ribosomal as well as nonribosomal) is recovered intact and in high yield. Fibroin messenger RNA (M,. 5.8 x 10”) isolated by this procedure is biologically active.
The most commonly used procedure for the isolation of nucleic acids from cells or tissues is the two-phase phenol extraction (l-3). Various modifications have been introduced to make phenol extraction a very efficient method, such as the use of detergents (SDS),’ proteases, phenol-chloroform mixtures, and phase reextractions with buffers of different pH (for review see Brawerman (4). Although efficient, these extractions are sometimes laborious and even hazardous, especially when hot phenol mixtures are used. We have taken advantage of the observations published by Wilcockson (5.6) and Wilcockson and Hull (7) on the properties of ethanolic sodium perchlorate to develop a procedure which can be used to isolate RNA from a variety of sources. The procedure is simple, efficient, and less hazardous than phenol extraction. In addition, it lends itself easily to the extraction of microscale samples. MATERIALS Proteinase Laboratories
solutions (3.5 mg/ml) were stored at -80°C. Sodium perchlorate (Analar grade) was obtained from BDH Chemicals. Stock solutions of sodium perchlorate in water were filtered through IO-pm Teflon filters (Millipore) and adjusted to pH 6-7. Ethanolic sodium perchlorate reagent (EPR) was prepared as described by Wilcockson (6) by mixing 1 vol of saturated salt in water with 4 vol of saturated salt in ethanol. Phenol was redistilled and stored under Hz0 at 4°C in the dark. Protc~inasr K digestion. Posterior silk glands from one or two fifth-instar larvae (0.25-0.50 g wet wt) were homogenized by seven strokes of a loose-fitting Dounce homogenizer in 6 ml of a buffer consisting of 2.4% SDS, 0.1 M NaCl, 7.5 mM EDTA, 20 Fglml PVS, 25 mM Tris-Cl, pH 7.35, and 350 pg/ml proteinase K. The homogenate was incubated (with magnetic stirring) for 12 min at 35-37°C. The same digestion conditions were used for dog pancreas and spinach leaves, except that in this case disruption was achieved by grinding in a mortar under liquid nitrogen. About 0.5 g of cold powdered tissue was added to 6 ml of digestion buffer. Proteinase K digestion of tissue culture cells was simply done by mixing 1 vol of concentrated cell suspension
AND METHODS
K was obtained from (E. Merck, Darmstadt).
E. M. Stock
’ Abbreviations
used: SDS, sodium dodecyl sulfate; sodium perchlorate (reagent): Mes. 2(N-morpholino)ethanesulfonic acid; PVS. polyvinyl sulfate.
EPR. ethanolic
0003-2697/79/130116-07$02.00/O Copyright ? 1979 by Academic Press. Inc. All nghfs of rsproduct~on m any term reserved.
116
RAPID
l.SOLATION
with 1 vol of twice-concentrated digestion buffer. The total volume of digestion mixture was about 6 ml for 1.0 x 10H cells. Sodium perchlorate extraction. To 6 ml of proteinase K-SDS lysate was added 4..0 ml of 3.5 M sodium perchlorate. The mixture was incubated for 3-4 min at 50°C (with agitation) until it looked clear. For silk glands, spinach leaves, or other tissues containing insoluble fibrous material, a filtration step was done at this point. The warm lysate was placed in Millipore cent,rifuge tubes (Catalog No. Xx6202550). containing a Mitex-LC lo-pm Teflon filter and a glassfiber prefilter, and centrifuged for !i min at 2900 rpm (15OOg)in a table-top centrifuge a 37°C. This centrifugation step shears DNA and reduces the viscosity of the lysate. The cleared lysate (nucleic acid concentration 2250 &ml) was placed in a 50-ml Corer; tube, and 40 ml of EPR reagent was added (EPR reagent is kept at room temperature:; 5 ml is added initially, with swirling, followed by another 35 ml, and thorough mixing). The solution was incubated in a water bath at 5°C for 30 min; then the precipitated nucleic acids were collected by centrifugation at 3800 rpm (3000g) for 10 min (5°C). The nucleic acid pellet was dissolved in 15 ml of 25 mM Tris-Cl, pH 7.35, 0.2% SDS, 1 mM EDTA, and nucleic acids were reprecipitated by adding NaCl to 0.18 M and 0.6 vol of isopropanol, and incubating at -20°C for 4 h. A final reprecipitation step with ethanol was done to remove residual perchlorate. Pllc~rlol-cllloro~~~ml extraction. Phenol extractions were performed with or without a prior proteinase K digestion step, as specified for each experiment. The SDS lysate (or SDS + proteinase K) was extracted by shaking for 10 min at 25°C with an equal volume of phenol-chloroform-isoarnyl alcohol (1: 1:0.02). The aqueous phase was saved, and the organic phase was reextracted with 1 vol of0.5’% SDS, 100I’IIM Tris-Cl. pH 9.0. After shaking for 5 min at 37°C the aqueous phase was harvested by centrifu-
117
OF RNA
gation and pooled with the first aqueous phase. The combined aqueous phases were reextracted with an equal volume of phenol-chloroform-isoamyl alcohol. The final aqueous phase was adjusted to 0.2 M NaCl and nucleic acids were precipitated with 2.5 vol of ethanol. An additional precipitation step with isopropanol was sometimes done for purposes of comparison with the perchlorate protocol. DNase digestion. Pancreatic deoxyribonuclease I (Worthington DPFF) was treated with iodoacetate to remove traces of RNase activity using the methods of Zimmerman and Sandeen (8). Nucleic acids were dissolved (1.4 mgiml in 10 mM Mes, pH 5.5, 0.1 M NaCl, 5 mM MgCIz, 10 pg/ml PVS). DNase was added to 15 pgiml and the material was incubated 20 min at 30°C. NaCl was added to 0.2 M and RNA was precipitated with 0.6 vol of isopropanol, followed by a reprecipitation with ethanol. Gel turing
elec,trophorrsis conditions.
under
Jidly
dena-
Gels containing 0.6% agarose and 1.6% acrylamide were cast in the presence of 52% formamide, 40 mM triethanolamine, 1 M formaldehyde, 1.3 mM EDTA, pH 7.5. The agarose was allowed to polymerize first by cooling the gel to 4°C for 1 h. The gel was then placed at room temperature overnight to allow for complete polymerization of acrylamide. Prior to loading, the samples were denatured in gel buffer containing 58% formamide and I .5 M formaldehyde by heating for 5 min at 60°C. The electrode buffer, which was recirculated, consisted of 40 mM triethanolamine. 2.5 mM NaCI. 1.25 mM disodium EDTA. pH 7.5. Electrophoresis was carried out for 22 h at 2.7 V/cm (25°C). The gels were stained with Stains-all (9). RESULTS AND DISCUSSION
Wilcockson has described methods for the isolation of nucleic acids from a number of sources by ethanol precipitation in the presence of sodium perchlorate (6). The
118
LIZARD1
AND
method works on the principle that proteins which normally precipitate in the presence of ethanol will remain in solution if sodium perchlorate is present in high concentrations. When we attempted to use this method for the isolation of RNA from the posterior silk gland of Botnbyx tnori we obtained lower yields than by phenol extraction, as well as significant protein contamination. In an effort to overcome the limitations of the Wilcockson method, we modified the procedure in a number of ways (see Materials and Methods). The use of a preliminary proteinase K digestion (IO, 11) resulted in greatly improved solubilization of the tissue by the SDS-sodium perchlorate solution. For the specific case of silk glands, it was found advantageous to centrifuge the SDS-perchlorate lysate through a IO-pm Teflon filter. This step removed fibrous tracheolar material which is resistant to protease and tends to float upon centrifugation. The inclusion of this filtration step results in significant shear breakage of DNA, but high molecular weight RNA remains intact as will be shown below. The addition of an isopropanol precipitation step reduced protein contamination and made the final nucleic acid pellet easier to redissolve.
ENGEl.BERG
The new perchlorate extraction protocol described in this paper gives excellent results when used for the extraction of silk gland RNA. To investigate whether the method had more general applicability, we used it to isolate nucleic acids from a variety of other sources. For comparison, we used a conventional phenol-chloroform extraction protocol, and a proteinase K + phenol procedure (see Materials and Methods). Table 1 shows that in terms of absolute yield the perchlorate method is comparable or in some cases superior to phenol extraction. Protein contamination is low, as suggested by the high ALR,~,2X,, ratios. A striking feature of the data in this table is the relatively poor yields obtained with phenol extraction in the absence of protease treatment. The value of the isopropanol precipitation step was especially evident in the case of the spinach extraction. Nucleic acid pellets from spinach were free from yellow-green pigments only if the extraction protocol included an isopropanol precipitation. To quantitate more accurately the level of protein contamination in RNA extracted using the perchlorate method the following experiment was performed. A postribosomal supernatant of CIIIcrtt~qdomottris cells labeled with [:‘“S]sulfate was mixed with
TABLE YIELDS
Source
01
NUCLEIC
Ac ID EXI
Proteinase K + perchlorate extraction
of material
Posterior silk glands (one fifth-instar larva) HeLa cells’ (1.6 x IO’) Dog pancreas (0.6 g) Spinach leaves (I gt Dog pancreas microsomes t -200 A,,;,, units) ” Values represent ratios. ’ These extractions ” The extractions Methods.
VARIOUS
I
Y6.9 84.9 213.0 20.4
the average
RACTION
METHOEW
Phenol extraction
Proteinase K + phenol extraction
(2.11)” (2.00) (2.01)” (2.00)”
88.3 (2.02) 79.6 (1.91) 90.5 ( 1 .X9) -
163.0 (2.17)
137.0 (2.101
AZ,;,, for
two determinations.
The numbers
in parentheses
included a filtration step, as described under Materials and Methods. of HeLa cell RNA included a DNase treatment step as specified under
97.3 (2. IO) 86.4 ( I .94) 14.5 ( I .YS)
are the A,,,,A,,,,
Materials
and
RAPID
ISOLATION
OF
RNA
119
perchlorate or phenol extraction. Figure 1A shows fractions from posterior silk :‘“S RADIOACTIVITY IN RNA PELLETS” glands extracted using either phenol (lane 1) ““S after NaOH or perchlorate (lane 2). It is clear that the + TCA” yield and size integrity of fibroin mRNA is Extraction method A ?M (cpm) the samefor both methods. Figure I B shows high molecular weight RNA from HeLa Proteinase K cells extracted with phenol or perchlorate + sodium perchlorate 64. I 1.72 r IO” Proteinase K (lanes 1 and 2). Lanes 3 and 4 show size + phenol-chloroform 65.3 2.23 z IO’ markers. Again it is clear that perchlorate extraction yields RNA of equivalent size ” The initial level of radioactivity in each proteinase integrity to phenol extraction, as demonK lysate was 4 x lo* cpm. All numbers in the table represent the average of two determinations. strated by the excellent recovery of 45 S ’ Sample5 from dried RNA pellets were dissolved in rRNA precursor. 0.1 M NaOH and incubated for 15 min at 37°C to deAnother critical question regarding RNA stroy aminoacylated tRNA. The material was then preextraction methods concerns the recovery cipitated with X’G trichloroacetic acid (TCA) (30 min. of hnRNA. It has been reported by a number 0°C). collected on glass filters. washed with 7:; TCA and ether. dried. and counted. of investigators (for review see (4)) that the use of certain phenol extraction protocols results in preferential losses of polyade0.7 g of rat liver and subjected to pronylated or “DNA-like” RNA. We have teinase K digestion, followed by extraction used a variety of phenol extraction protocols with either sodium perchlorate or phenolto extract pulse-labeled RNA from silk chloroform (see Materials and Methods). glands and have found considerable variAliquots of the final nucleic acid pellets ation in the yield of fibroin mRNA and were counted as described in the legend to hnRNA. Apparent losses were observed Table 2. The table shows that the level of with phenol extraction protocols in which contamination by ‘“S-labeled material is the protease-SDS digestion was omitted, lower for the perchlorate-extracted mateor when phenol alone (without chloroform) rial. We should point out that omission of was used. Among the phenol protocols we the proteinase K digestion step, or, alternaused, proteinase K/SDS + phenolichlorotively, the use of sodium chloride instlead of form gave the best yield of pulse-labeled sodium perchlorate, results in significantly material. higher levels of residual :rr,S contamination We therefore decided to compare the (not shown). Thus, both the proteolysis perchlorate method with the most efficient step and the presence of the perchlorate ion of the phenol methods in terms of the are essential for optimal purity. recovery of pulse-labeled RNA. DoubleWe have examined the structural integrity labeled RNA was extracted using either of the RNA obtained using the perchlorate method, as described in the legend to procedure. A rather critical test of structural Fig. 2. A preliminary sucrose gradient integrity is provided by a very large mRNA fractionation step was done to select RNA such as fibroin mRNA (molecular weight larger than 20 S, in order to facilitate further approximately 5.8 x IO”; see(12)). We have analysis of heavy hnRNA. The heavy RNA found that the perchlorate extraction was heat denatured in 60% formamide, consistently yields intact fibroin mRNA as and analyzed in formamide gradients as assayed by gel electrophoresis. Figure 1 shown in Fig. 2. Under these conditions the shows a denaturing gel analysis of various 28 S rRNA of B. rm~i splits into two halves RNA fractions extracted using (either which sediment at about 18 S. The lower TABLE
2
120
LIZARD1
AND
1
A
-e
ENGELBERG
2 ,*I -w-, _ ““s 4 I.
3
-f
I
-38s
mRNA
-185
f3
FIG. 1. Gel electrophoretic analysis of RNA fractions extracted with various methods. (A) Posterior silk gland RNA extracted with proteinase K + phenol-chloroform (lane 1) or proteinase K + perchlorate procedure (lane 2). Gel electrophoresis in formaldehyde gels was as described by Lizardi it N/. (IO). (B) Lane 1: HeLa ceil RNA extracted with phenol-chloroform and enriched for material larger than 20 S by sucrose gradient centrifugation. Lane 2: HeLa cell RNA extracted by perchlorate method; otherwise treated as in lane 1. Lane 3: fibroin mRNA marker. Lane 4: Total RNA from mouse 3T6 cells. Fully denaturing gel electrophoresis in formamide-formaldehyde gels was as described under Materials and Methods. The faint 16 S band in the HeLa RNA preparation is a contaminant derived from Eschcric~l~iu c,o/i tRNA used as carrier during the final alcohol precipitation steps.
quadrant of Fig. 2 (open circles) shows the pattern of radioactivity of stable RNA, which was found to be identical for both phenol- and perchlorate-extracted material. The major peak sedimenting heavier than 40 S is fibroin mRNA, which accumulates during the 24-h “2P-labeling period. The upper part of Fig. 2 shows the (“H/“‘P) ratios for each fraction of two separate gradients containing perchlorateor phenol-extracted
material, respectively. The isotope ratio is a measure of the efficiency of extraction of pulse-labeled [“HIRNA. As expected, the highest ratios are present at 38 S (ribosomal RNA precursor) and at 50-65 S (large hnRNA). We know from previous work (13) that about one-half of this 50-65 S hnRNA binds to poly(U)-Sepharose and therefore contains poly(A). The profile of isotope ratios in Fig. 2 shows that the perchlorate
RAPID
Fraction
ISOLATION
number
FIG. 2. Sucrose gradient RNA extracted with phenol
analysis of double-labeled or perchlorate. Each oftwo
silkworms
labeled
(fifth
instar)
was
with
0.2 mCi
[:l”P]-
orthophosphate for 24 h. and with 0.9 mCi of [“HIuridine for 15 min prior to extraction. A proteinase K-SDS lysate of posterior silk glands was prepared, and equal aliquots were extracted using either the perchlorate larger than
or
preparative layered
sucrosegradient. analytical
made
over
up in 60c+
EDTA.O.l’-; in a Beckman was The
the phenol-chloroform 20 S wab isolated
heated protiles
formamide.
IO mM Tris,
SDS.andrunfor SW 17. I rotor. to 65°C for 3 min of “P radioactivity
of perchlorate
method. RNA a preliminary
The heavy RNA formamide gradients
phenoland perchlorate-extracted only the phenol ,“P profile has quadrant shows the (,‘HYP) gradients
by
pH
was then (5-20’;) 7.5,
18 hat26,500rpm(30”C) Prior to loading the in 601? formamide were identical been ratios
I rnh< RNA
buffer. for both
RNA. and therefore plotted. The upper for two separate
or phenol-extracted
RNA,
extraction is as good if not better than the phenol-chloroform extraction in terms of the yield of 50-65 S pulse-labeled hnlRNA. Similar experiments (not shown) were done to compare the yields of smaller RNAs, and again the perchlorate and phenolchloroform methods were found to be equivalent. In fact, on theoretical grounds one would predict that the perchlorate method is less likely to produce selective losses because it does not involve the separation of liquid phases.
OF
121
RNA
Another relevant point concerns the biological activity of RNA isolated using the perchlorate method described in this paper. Fibroin messenger RNA has been isolated using this method and translated in a reticulocyte mRNA-dependent cellfree system (14). The translational activity of perchlorate-extracted mRNA has been found to be indistinguishable from that of phenol-extracted mRNA (Lizardi cf al., manuscript in preparation). Similar results have been found by Scheele and Blackburn (personal communication). who have translated dog pancreas mRNA extracted with the perchlorate method. Finally we would like to point out that the perchlorate protocol described in the Materials and Methods section can be simplified when used for certain applications. In those cases where clear lysates are obtained after mixing in the presence of SDS, proteinase K, and sodium perchlorate, a filtration step is not necessary unless one wishes to shear DNA. Clear lysates are easily obtained with extracts of tissue culture cells, RNA viruses, polysomes, ribonucleoprotein particles, rough microsomes, etc. For these applications the method lends itself easily to the extraction of microscale samples. For example, we have extracted RNA from very small microsomal pellets using a total extraction volume of 40 ~1. Spectrophotometric determinations of RNA concentration can be done after only one precipitation step because perchlorate does not absorb at 260 nm. When extracting RNA-rich material such as polysomes or other ribonucleoprotein particles one can omit the proteinase K digestion; the protocol is thus reduced to solubilization in SDSiperchlorate at 50°C and precipitation with ethanoliperchlorate followed by reprecipitation with isopropanol or ethanol. ACKNOWLEDGMENTS We wish to thank K. Matlln for a gift of “S-labeled crrItr~,r~till~llr,rlrr.\ . W. Jelinek for a gift of HeLa cells. and V. Mahdavi and G. Piperno for critical reading
122
LIZARD1
AND
of the manuscript. This work was supported by Grant GM-22865 from the U. S. Public Health Service.
REFERENCES I. Gierer, A., and Schramm, G. ( 1956)Z. iV~~t~r~/~~vsc~/r. B 11, 138. 2. Kirby, K. S. (1967) ilt Techniques in Protein Biosynthesis (Campbell, P. N., and Sargent, J. R.. eds.), Vol. 1. pp. 265-297. Academic Press, New York. 3. Kirby. K. S. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K.. eds.). Vol. 12B. pp. 87-99, Academic Press, New York. 4. Brdwerman. G. (1973) in Methods in Cell Biology (Prescott. D. M.. ed.). Vol. 7. pp. l-22, Academic Press, New York. 5. Wilcockson, J. (1973) Bioclrrm. J. 135, 559-561.
ENGELBERG 6. Wilcockson, J. ( 1975) A!rul. B/oc,/rchr. 66, 64-68. 7. Wilcockson. J.. and Hull. R. (1974) J. (;(,!I. l’in~l. 23. 107-111. 8. Zimmerman. S. B.. and Sandeen, G. t 1966) ALIBI/. Bicv //~‘/11. 14, 2699277. 9. Dahlberg. A. E.. Dingman. C. W.. and Peacock. A. C. (196Yl.I. 1f<~/. Rio/. 41. 139-147. IO. Wiegers. U.. and HilL. H. (1972) FEB.5 Lc/!. 23, 77-82. I I. Hilz, H.. Wiegers. U.. and Adamietz. P. (1975) Eur. .I. Bicu~hrm. 56, lO3- 108. 12. Lizardi, P. M.. Williamson. R.. and Brown. D. D. (1975) Cell 4, 199-205. 13. Lizardi, P. M. (1976) i/t Progress in Nucleic Acid Research and Molecular Biology (Cohn, W. E.. and Volkin. E., eds.). Vol. 19, pp. 301-312, Academic Press, New York. 14. Pelham. H. R. B.. and Jackson. R. J. (1976) Eur. J. Bioc,irt,m. 67, 247-256.
BIOC HtMlSl
RY 98.
1 Ih-
I??
( 1979)
Rapid Isolation of RNA Using Proteinase K and Sodium Pet-chlorate PAUL M. LIZARDI The
Rdefdkr
AND ALAN ENGELBERG
lJt~i~wsit,v.
Received
Nm.
February
k;w~.
Nru.
l’ori
10021
28, 1979
A simple. efficient procedure for the isolation of cellular nucleic acids is described. It combines the use of sodium dodecyl sulfate. proteinase K, sodium perchlorate, and isopropanol precipitation. The yields and purity of RNA extracted from a variety of sources are comparable or superior to those obtained by phenol extraction. High molecular weight RNA (ribosomal as well as nonribosomal) is recovered intact and in high yield. Fibroin messenger RNA (M,. 5.8 x 10”) isolated by this procedure is biologically active.
The most commonly used procedure for the isolation of nucleic acids from cells or tissues is the two-phase phenol extraction (l-3). Various modifications have been introduced to make phenol extraction a very efficient method, such as the use of detergents (SDS),’ proteases, phenol-chloroform mixtures, and phase reextractions with buffers of different pH (for review see Brawerman (4). Although efficient, these extractions are sometimes laborious and even hazardous, especially when hot phenol mixtures are used. We have taken advantage of the observations published by Wilcockson (5.6) and Wilcockson and Hull (7) on the properties of ethanolic sodium perchlorate to develop a procedure which can be used to isolate RNA from a variety of sources. The procedure is simple, efficient, and less hazardous than phenol extraction. In addition, it lends itself easily to the extraction of microscale samples. MATERIALS Proteinase Laboratories
solutions (3.5 mg/ml) were stored at -80°C. Sodium perchlorate (Analar grade) was obtained from BDH Chemicals. Stock solutions of sodium perchlorate in water were filtered through IO-pm Teflon filters (Millipore) and adjusted to pH 6-7. Ethanolic sodium perchlorate reagent (EPR) was prepared as described by Wilcockson (6) by mixing 1 vol of saturated salt in water with 4 vol of saturated salt in ethanol. Phenol was redistilled and stored under Hz0 at 4°C in the dark. Protc~inasr K digestion. Posterior silk glands from one or two fifth-instar larvae (0.25-0.50 g wet wt) were homogenized by seven strokes of a loose-fitting Dounce homogenizer in 6 ml of a buffer consisting of 2.4% SDS, 0.1 M NaCl, 7.5 mM EDTA, 20 Fglml PVS, 25 mM Tris-Cl, pH 7.35, and 350 pg/ml proteinase K. The homogenate was incubated (with magnetic stirring) for 12 min at 35-37°C. The same digestion conditions were used for dog pancreas and spinach leaves, except that in this case disruption was achieved by grinding in a mortar under liquid nitrogen. About 0.5 g of cold powdered tissue was added to 6 ml of digestion buffer. Proteinase K digestion of tissue culture cells was simply done by mixing 1 vol of concentrated cell suspension
AND METHODS
K was obtained from (E. Merck, Darmstadt).
E. M. Stock
’ Abbreviations
used: SDS, sodium dodecyl sulfate; sodium perchlorate (reagent): Mes. 2(N-morpholino)ethanesulfonic acid; PVS. polyvinyl sulfate.
EPR. ethanolic
0003-2697/79/130116-07$02.00/O Copyright ? 1979 by Academic Press. Inc. All nghfs of rsproduct~on m any term reserved.
116
RAPID
l.SOLATION
with 1 vol of twice-concentrated digestion buffer. The total volume of digestion mixture was about 6 ml for 1.0 x 10H cells. Sodium perchlorate extraction. To 6 ml of proteinase K-SDS lysate was added 4..0 ml of 3.5 M sodium perchlorate. The mixture was incubated for 3-4 min at 50°C (with agitation) until it looked clear. For silk glands, spinach leaves, or other tissues containing insoluble fibrous material, a filtration step was done at this point. The warm lysate was placed in Millipore cent,rifuge tubes (Catalog No. Xx6202550). containing a Mitex-LC lo-pm Teflon filter and a glassfiber prefilter, and centrifuged for !i min at 2900 rpm (15OOg)in a table-top centrifuge a 37°C. This centrifugation step shears DNA and reduces the viscosity of the lysate. The cleared lysate (nucleic acid concentration 2250 &ml) was placed in a 50-ml Corer; tube, and 40 ml of EPR reagent was added (EPR reagent is kept at room temperature:; 5 ml is added initially, with swirling, followed by another 35 ml, and thorough mixing). The solution was incubated in a water bath at 5°C for 30 min; then the precipitated nucleic acids were collected by centrifugation at 3800 rpm (3000g) for 10 min (5°C). The nucleic acid pellet was dissolved in 15 ml of 25 mM Tris-Cl, pH 7.35, 0.2% SDS, 1 mM EDTA, and nucleic acids were reprecipitated by adding NaCl to 0.18 M and 0.6 vol of isopropanol, and incubating at -20°C for 4 h. A final reprecipitation step with ethanol was done to remove residual perchlorate. Pllc~rlol-cllloro~~~ml extraction. Phenol extractions were performed with or without a prior proteinase K digestion step, as specified for each experiment. The SDS lysate (or SDS + proteinase K) was extracted by shaking for 10 min at 25°C with an equal volume of phenol-chloroform-isoarnyl alcohol (1: 1:0.02). The aqueous phase was saved, and the organic phase was reextracted with 1 vol of0.5’% SDS, 100I’IIM Tris-Cl. pH 9.0. After shaking for 5 min at 37°C the aqueous phase was harvested by centrifu-
117
OF RNA
gation and pooled with the first aqueous phase. The combined aqueous phases were reextracted with an equal volume of phenol-chloroform-isoamyl alcohol. The final aqueous phase was adjusted to 0.2 M NaCl and nucleic acids were precipitated with 2.5 vol of ethanol. An additional precipitation step with isopropanol was sometimes done for purposes of comparison with the perchlorate protocol. DNase digestion. Pancreatic deoxyribonuclease I (Worthington DPFF) was treated with iodoacetate to remove traces of RNase activity using the methods of Zimmerman and Sandeen (8). Nucleic acids were dissolved (1.4 mgiml in 10 mM Mes, pH 5.5, 0.1 M NaCl, 5 mM MgCIz, 10 pg/ml PVS). DNase was added to 15 pgiml and the material was incubated 20 min at 30°C. NaCl was added to 0.2 M and RNA was precipitated with 0.6 vol of isopropanol, followed by a reprecipitation with ethanol. Gel turing
elec,trophorrsis conditions.
under
Jidly
dena-
Gels containing 0.6% agarose and 1.6% acrylamide were cast in the presence of 52% formamide, 40 mM triethanolamine, 1 M formaldehyde, 1.3 mM EDTA, pH 7.5. The agarose was allowed to polymerize first by cooling the gel to 4°C for 1 h. The gel was then placed at room temperature overnight to allow for complete polymerization of acrylamide. Prior to loading, the samples were denatured in gel buffer containing 58% formamide and I .5 M formaldehyde by heating for 5 min at 60°C. The electrode buffer, which was recirculated, consisted of 40 mM triethanolamine. 2.5 mM NaCI. 1.25 mM disodium EDTA. pH 7.5. Electrophoresis was carried out for 22 h at 2.7 V/cm (25°C). The gels were stained with Stains-all (9). RESULTS AND DISCUSSION
Wilcockson has described methods for the isolation of nucleic acids from a number of sources by ethanol precipitation in the presence of sodium perchlorate (6). The
118
LIZARD1
AND
method works on the principle that proteins which normally precipitate in the presence of ethanol will remain in solution if sodium perchlorate is present in high concentrations. When we attempted to use this method for the isolation of RNA from the posterior silk gland of Botnbyx tnori we obtained lower yields than by phenol extraction, as well as significant protein contamination. In an effort to overcome the limitations of the Wilcockson method, we modified the procedure in a number of ways (see Materials and Methods). The use of a preliminary proteinase K digestion (IO, 11) resulted in greatly improved solubilization of the tissue by the SDS-sodium perchlorate solution. For the specific case of silk glands, it was found advantageous to centrifuge the SDS-perchlorate lysate through a IO-pm Teflon filter. This step removed fibrous tracheolar material which is resistant to protease and tends to float upon centrifugation. The inclusion of this filtration step results in significant shear breakage of DNA, but high molecular weight RNA remains intact as will be shown below. The addition of an isopropanol precipitation step reduced protein contamination and made the final nucleic acid pellet easier to redissolve.
ENGEl.BERG
The new perchlorate extraction protocol described in this paper gives excellent results when used for the extraction of silk gland RNA. To investigate whether the method had more general applicability, we used it to isolate nucleic acids from a variety of other sources. For comparison, we used a conventional phenol-chloroform extraction protocol, and a proteinase K + phenol procedure (see Materials and Methods). Table 1 shows that in terms of absolute yield the perchlorate method is comparable or in some cases superior to phenol extraction. Protein contamination is low, as suggested by the high ALR,~,2X,, ratios. A striking feature of the data in this table is the relatively poor yields obtained with phenol extraction in the absence of protease treatment. The value of the isopropanol precipitation step was especially evident in the case of the spinach extraction. Nucleic acid pellets from spinach were free from yellow-green pigments only if the extraction protocol included an isopropanol precipitation. To quantitate more accurately the level of protein contamination in RNA extracted using the perchlorate method the following experiment was performed. A postribosomal supernatant of CIIIcrtt~qdomottris cells labeled with [:‘“S]sulfate was mixed with
TABLE YIELDS
Source
01
NUCLEIC
Ac ID EXI
Proteinase K + perchlorate extraction
of material
Posterior silk glands (one fifth-instar larva) HeLa cells’ (1.6 x IO’) Dog pancreas (0.6 g) Spinach leaves (I gt Dog pancreas microsomes t -200 A,,;,, units) ” Values represent ratios. ’ These extractions ” The extractions Methods.
VARIOUS
I
Y6.9 84.9 213.0 20.4
the average
RACTION
METHOEW
Phenol extraction
Proteinase K + phenol extraction
(2.11)” (2.00) (2.01)” (2.00)”
88.3 (2.02) 79.6 (1.91) 90.5 ( 1 .X9) -
163.0 (2.17)
137.0 (2.101
AZ,;,, for
two determinations.
The numbers
in parentheses
included a filtration step, as described under Materials and Methods. of HeLa cell RNA included a DNase treatment step as specified under
97.3 (2. IO) 86.4 ( I .94) 14.5 ( I .YS)
are the A,,,,A,,,,
Materials
and
RAPID
ISOLATION
OF
RNA
119
perchlorate or phenol extraction. Figure 1A shows fractions from posterior silk :‘“S RADIOACTIVITY IN RNA PELLETS” glands extracted using either phenol (lane 1) ““S after NaOH or perchlorate (lane 2). It is clear that the + TCA” yield and size integrity of fibroin mRNA is Extraction method A ?M (cpm) the samefor both methods. Figure I B shows high molecular weight RNA from HeLa Proteinase K cells extracted with phenol or perchlorate + sodium perchlorate 64. I 1.72 r IO” Proteinase K (lanes 1 and 2). Lanes 3 and 4 show size + phenol-chloroform 65.3 2.23 z IO’ markers. Again it is clear that perchlorate extraction yields RNA of equivalent size ” The initial level of radioactivity in each proteinase integrity to phenol extraction, as demonK lysate was 4 x lo* cpm. All numbers in the table represent the average of two determinations. strated by the excellent recovery of 45 S ’ Sample5 from dried RNA pellets were dissolved in rRNA precursor. 0.1 M NaOH and incubated for 15 min at 37°C to deAnother critical question regarding RNA stroy aminoacylated tRNA. The material was then preextraction methods concerns the recovery cipitated with X’G trichloroacetic acid (TCA) (30 min. of hnRNA. It has been reported by a number 0°C). collected on glass filters. washed with 7:; TCA and ether. dried. and counted. of investigators (for review see (4)) that the use of certain phenol extraction protocols results in preferential losses of polyade0.7 g of rat liver and subjected to pronylated or “DNA-like” RNA. We have teinase K digestion, followed by extraction used a variety of phenol extraction protocols with either sodium perchlorate or phenolto extract pulse-labeled RNA from silk chloroform (see Materials and Methods). glands and have found considerable variAliquots of the final nucleic acid pellets ation in the yield of fibroin mRNA and were counted as described in the legend to hnRNA. Apparent losses were observed Table 2. The table shows that the level of with phenol extraction protocols in which contamination by ‘“S-labeled material is the protease-SDS digestion was omitted, lower for the perchlorate-extracted mateor when phenol alone (without chloroform) rial. We should point out that omission of was used. Among the phenol protocols we the proteinase K digestion step, or, alternaused, proteinase K/SDS + phenolichlorotively, the use of sodium chloride instlead of form gave the best yield of pulse-labeled sodium perchlorate, results in significantly material. higher levels of residual :rr,S contamination We therefore decided to compare the (not shown). Thus, both the proteolysis perchlorate method with the most efficient step and the presence of the perchlorate ion of the phenol methods in terms of the are essential for optimal purity. recovery of pulse-labeled RNA. DoubleWe have examined the structural integrity labeled RNA was extracted using either of the RNA obtained using the perchlorate method, as described in the legend to procedure. A rather critical test of structural Fig. 2. A preliminary sucrose gradient integrity is provided by a very large mRNA fractionation step was done to select RNA such as fibroin mRNA (molecular weight larger than 20 S, in order to facilitate further approximately 5.8 x IO”; see(12)). We have analysis of heavy hnRNA. The heavy RNA found that the perchlorate extraction was heat denatured in 60% formamide, consistently yields intact fibroin mRNA as and analyzed in formamide gradients as assayed by gel electrophoresis. Figure 1 shown in Fig. 2. Under these conditions the shows a denaturing gel analysis of various 28 S rRNA of B. rm~i splits into two halves RNA fractions extracted using (either which sediment at about 18 S. The lower TABLE
2
120
LIZARD1
AND
1
A
-e
ENGELBERG
2 ,*I -w-, _ ““s 4 I.
3
-f
I
-38s
mRNA
-185
f3
FIG. 1. Gel electrophoretic analysis of RNA fractions extracted with various methods. (A) Posterior silk gland RNA extracted with proteinase K + phenol-chloroform (lane 1) or proteinase K + perchlorate procedure (lane 2). Gel electrophoresis in formaldehyde gels was as described by Lizardi it N/. (IO). (B) Lane 1: HeLa ceil RNA extracted with phenol-chloroform and enriched for material larger than 20 S by sucrose gradient centrifugation. Lane 2: HeLa cell RNA extracted by perchlorate method; otherwise treated as in lane 1. Lane 3: fibroin mRNA marker. Lane 4: Total RNA from mouse 3T6 cells. Fully denaturing gel electrophoresis in formamide-formaldehyde gels was as described under Materials and Methods. The faint 16 S band in the HeLa RNA preparation is a contaminant derived from Eschcric~l~iu c,o/i tRNA used as carrier during the final alcohol precipitation steps.
quadrant of Fig. 2 (open circles) shows the pattern of radioactivity of stable RNA, which was found to be identical for both phenol- and perchlorate-extracted material. The major peak sedimenting heavier than 40 S is fibroin mRNA, which accumulates during the 24-h “2P-labeling period. The upper part of Fig. 2 shows the (“H/“‘P) ratios for each fraction of two separate gradients containing perchlorateor phenol-extracted
material, respectively. The isotope ratio is a measure of the efficiency of extraction of pulse-labeled [“HIRNA. As expected, the highest ratios are present at 38 S (ribosomal RNA precursor) and at 50-65 S (large hnRNA). We know from previous work (13) that about one-half of this 50-65 S hnRNA binds to poly(U)-Sepharose and therefore contains poly(A). The profile of isotope ratios in Fig. 2 shows that the perchlorate
RAPID
Fraction
ISOLATION
number
FIG. 2. Sucrose gradient RNA extracted with phenol
analysis of double-labeled or perchlorate. Each oftwo
silkworms
labeled
(fifth
instar)
was
with
0.2 mCi
[:l”P]-
orthophosphate for 24 h. and with 0.9 mCi of [“HIuridine for 15 min prior to extraction. A proteinase K-SDS lysate of posterior silk glands was prepared, and equal aliquots were extracted using either the perchlorate larger than
or
preparative layered
sucrosegradient. analytical
made
over
up in 60c+
EDTA.O.l’-; in a Beckman was The
the phenol-chloroform 20 S wab isolated
heated protiles
formamide.
IO mM Tris,
SDS.andrunfor SW 17. I rotor. to 65°C for 3 min of “P radioactivity
of perchlorate
method. RNA a preliminary
The heavy RNA formamide gradients
phenoland perchlorate-extracted only the phenol ,“P profile has quadrant shows the (,‘HYP) gradients
by
pH
was then (5-20’;) 7.5,
18 hat26,500rpm(30”C) Prior to loading the in 601? formamide were identical been ratios
I rnh< RNA
buffer. for both
RNA. and therefore plotted. The upper for two separate
or phenol-extracted
RNA,
extraction is as good if not better than the phenol-chloroform extraction in terms of the yield of 50-65 S pulse-labeled hnlRNA. Similar experiments (not shown) were done to compare the yields of smaller RNAs, and again the perchlorate and phenolchloroform methods were found to be equivalent. In fact, on theoretical grounds one would predict that the perchlorate method is less likely to produce selective losses because it does not involve the separation of liquid phases.
OF
121
RNA
Another relevant point concerns the biological activity of RNA isolated using the perchlorate method described in this paper. Fibroin messenger RNA has been isolated using this method and translated in a reticulocyte mRNA-dependent cellfree system (14). The translational activity of perchlorate-extracted mRNA has been found to be indistinguishable from that of phenol-extracted mRNA (Lizardi cf al., manuscript in preparation). Similar results have been found by Scheele and Blackburn (personal communication). who have translated dog pancreas mRNA extracted with the perchlorate method. Finally we would like to point out that the perchlorate protocol described in the Materials and Methods section can be simplified when used for certain applications. In those cases where clear lysates are obtained after mixing in the presence of SDS, proteinase K, and sodium perchlorate, a filtration step is not necessary unless one wishes to shear DNA. Clear lysates are easily obtained with extracts of tissue culture cells, RNA viruses, polysomes, ribonucleoprotein particles, rough microsomes, etc. For these applications the method lends itself easily to the extraction of microscale samples. For example, we have extracted RNA from very small microsomal pellets using a total extraction volume of 40 ~1. Spectrophotometric determinations of RNA concentration can be done after only one precipitation step because perchlorate does not absorb at 260 nm. When extracting RNA-rich material such as polysomes or other ribonucleoprotein particles one can omit the proteinase K digestion; the protocol is thus reduced to solubilization in SDSiperchlorate at 50°C and precipitation with ethanoliperchlorate followed by reprecipitation with isopropanol or ethanol. ACKNOWLEDGMENTS We wish to thank K. Matlln for a gift of “S-labeled crrItr~,r~till~llr,rlrr.\ . W. Jelinek for a gift of HeLa cells. and V. Mahdavi and G. Piperno for critical reading
122
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of the manuscript. This work was supported by Grant GM-22865 from the U. S. Public Health Service.
REFERENCES I. Gierer, A., and Schramm, G. ( 1956)Z. iV~~t~r~/~~vsc~/r. B 11, 138. 2. Kirby, K. S. (1967) ilt Techniques in Protein Biosynthesis (Campbell, P. N., and Sargent, J. R.. eds.), Vol. 1. pp. 265-297. Academic Press, New York. 3. Kirby. K. S. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K.. eds.). Vol. 12B. pp. 87-99, Academic Press, New York. 4. Brdwerman. G. (1973) in Methods in Cell Biology (Prescott. D. M.. ed.). Vol. 7. pp. l-22, Academic Press, New York. 5. Wilcockson, J. (1973) Bioclrrm. J. 135, 559-561.
ENGELBERG 6. Wilcockson, J. ( 1975) A!rul. B/oc,/rchr. 66, 64-68. 7. Wilcockson. J.. and Hull. R. (1974) J. (;(,!I. l’in~l. 23. 107-111. 8. Zimmerman. S. B.. and Sandeen, G. t 1966) ALIBI/. Bicv //~‘/11. 14, 2699277. 9. Dahlberg. A. E.. Dingman. C. W.. and Peacock. A. C. (196Yl.I. 1f<~/. Rio/. 41. 139-147. IO. Wiegers. U.. and HilL. H. (1972) FEB.5 Lc/!. 23, 77-82. I I. Hilz, H.. Wiegers. U.. and Adamietz. P. (1975) Eur. .I. Bicu~hrm. 56, lO3- 108. 12. Lizardi, P. M.. Williamson. R.. and Brown. D. D. (1975) Cell 4, 199-205. 13. Lizardi, P. M. (1976) i/t Progress in Nucleic Acid Research and Molecular Biology (Cohn, W. E.. and Volkin. E., eds.). Vol. 19, pp. 301-312, Academic Press, New York. 14. Pelham. H. R. B.. and Jackson. R. J. (1976) Eur. J. Bioc,irt,m. 67, 247-256.