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Extraction of High Molecular Weight RNA and DNA from Cultured Mammalian Cells
[3] EXTRACTION OF R N A AND D N A FROM MAMMALIAN CELLS
37
[3] Extraction of High Molecular Weight RNA and DNA from Cultured Mammalian Cells By H. C. BIRNBOIM
Introduction Although a large number of techniques have been described for the isolation of pure high molecular weight RNA and DNA from mammalian cells, no method is universally applicable because of different problems encountered with different cell and tissue types and different uses of the purified nucleic acids. In general, it is difficult to prepare both RNA and high molecular weight DNA from the same extract,1,2 because each presents separate difficulties. The most common problem encountered in the isolation of intact RNA is the presence of stable ribonucleases inside and outside of cells. Because RNA is single stranded, one cut by a ribonuclease may destroy its usefulness. Thus, effective ribonuclease inhibitors are needed from the moment of initial cell lysis. RNA is not shear sensitive but it is very alkali labile, so the pH must be controlled at the pH of maximal stability (6.5-7.0), particularly if it is to be heated. Other factors need to be considered to isolate high molecular weight DNA. Deoxyribonucleases are less of a problem because, compared to ribonucleases, they are generally more labile and inhibitable by chelators. Furthermore, DNA is a duplex molecule, so that cleavage of one of the two strands usually does not destroy its experimental usefulness. Its high viscosity makes it difficult to redissolve after alcohol precipita tion and it is sensitive to degradation by shearing. Preparation of intact, chromosome-size pieces of DNA requires special procedures such as embedding cells in agarose,3 which will not be considered here. The present chapter describes methods for the isolation of highly purified RNA and DNA in near-quantitative yield from small numbers of cultured mammalian cells and white blood cells. These nucleic acids are suitable for Southern and Northern transfers and for other applications. The RNA extraction procedure is slightly modified from one described earlier.4
1
F. D. Kury, C. Schneeberger, G. Sliutz, E. Kubista, H. Salzer, M. Medl, S. Leodolter, H. Swoboda, R. Zeillinger, and J. Spona, Oncogene 5, 1403 (1990). 2 J. Karlinsey, G. Stamatoyannopoulos, and T. Enver, Anal. Biochem. 180, 303 (1989). 3 D. C. Schwartz and C. R. Cantor, Cell {Cambridge, Mass.) 37, 67 (1984). 4 H. C. Birnboim, Nucleic Acids Res. 16, 1487 (1988). RECOMBINANT DNA METHODOLOGY II
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
38
I. ISOLATION, SYNTHESIS, AND DETECTION OF D N A AND R N A
Extraction of RNA The moment of initial disruption of a cell is the time that ribonucleases are released and can cause degradation of cellular RNA. Cell extraction should therefore be carried out in a solution containing strong denaturing agents and/or other inhibitors of ribonucleases. The solution should be at pH 6.5-7.0 (the pH of optimal stability for RNA). 5 Although guanidinium thiocyanate is useful as a denaturing agent and ribonuclease inhibitor, 6-10 a mixture of sodium dodecyl sulfate (SDS) and a low concentration of urea 11 is an effective inhibitor of potent leukocyte ribonucleases, which are more difficult to inhibit than pancreatic ribonuclease. 4 At the same time, this mixture effectively activates proteinase K.12 Once most of the protein is digested, nucleic acids can be concentrated by precipitation with alcohol. This strategy minimizes the volume of phenol required for subsequent extractions and improves recovery. DNA can be eliminated almost completely by precipitation of high molecular weight RNA with LiCl, obviating the need for ribonuclease-free deoxyribonuclease or buoy ant density sedimentation. Reagents For RNA Extraction and Denaturation RES-1: 0.5 M LiCl, 1 M urea, 1.0% SDS, 0.02 M sodium citrate, and 2.5 mMcyclohexanediaminetetraacetate (CDTA) (final pH 6.8). Filter before addition of SDS and store at room temperature Proteinase K: 5 mg/ml in water; store at - 2 0 ° Phenol/chloroform: 10 g phenol crystals (analytical grade) dissolved in 10 ml of chloroform. Store at room temperature, protected from light. The solution should be colorless. The use of recrystallized phenol is unnecessary Sodium acetate (pH 5.2) (2 M in sodium ion): For 100 ml of solution, 0.2 mol of sodium acetate is adjusted to pH 5.2 (as measured at 0.05 M) with acetic acid and made up to volume. Filter, autoclave, and store at room temperature over chloroform Acetic acid: 2.0 M 5
H. C. Birnboim, unpublished observation (1974). J. H. Chirgwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979). 7 G. Cathala, J. F. Savouret, B. Mendez, B. L. West, M. Karin, J. A. Martail, and J. D. Baxter, DNA 2, 329(1983). 8 S. A. Krawetz, J. C. States, and G. H. Dixon, J. Biochem. Biophys. Methods 12, 29 (1986). 9 P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987). 10 S. L. Berger and J. M. Chirgwin, this series, Vol. 180, p. 3. 11 R. Soeiro, M. H. Vaughan, and J. E. Darnell, J. Cell Biol. 36, 91 (1968). 12 H. Hilz, U. Wiegers, and P. Adamietz, Eur. J. Biochem. 56, 103 (1975). 6
[3] EXTRACTION OF R N A AND D N A FROM MAMMALIAN CELLS
39
LiCl/ethanol: Combine 3 vol of 5 M LiCl (which has been filtered and autoclaved) with 2 vol of 95% ethanol. Store at room temperature CCS: 1 mM sodium citrate, 1 mM CDTA, 0.1% SDS, adjusted to pH 6.8. Autoclave before addition of SDS. Filter and store at room tem perature FFP (formaldehyde/formamide/phosphate): FFP [100 μ,Ι 37% formalde hyde, 94 μ,Ι formamide, 6.6 μΐ 1 M sodium phosphate (pH 6.8)] is freshly prepared before use. RNA will be partially degraded during heating prior to Northern analysis if the pH is <6.0. Formamide tends to become acidic on standing and the highest grade of formamide available (e.g., Omnisolv grade from BDH Chemicals, Poole, En gland) should be used. It can be stored at 4° over mixed-bed resin. Formaldehyde may also be acidic. The pH of formamide, formalde hyde, or mixtures is conveniently monitored using 2 vol bromthymol blue (0.25 mg/ml in 0.5 mM NaOH) as a color indicator Where indicated, solutions are filtered through a 0.45-μ,πι membrane filter. Cyclohexanediamine tetraacetate is a chelator that, compared to ethylenediaminetetraacetate (EDTA), binds metal ions 100-1000 more strongly, is much more soluble in acid and alcohol, and is easier to prepare in concentrated solutions. It is available from Sigma Chemical Co. (St. Louis, MO). RNA Extraction Procedure for Cultured Cells Volumes given are for 3-5 x 106 cells grown as a monolayer in a 10-cm petri dish and can be readily scaled up or down according to the cell number and surface area. The dish is chilled on an aluminum plate in contact with ice. The culture medium is quickly removed by aspiration and the cell layer washed twice with cold balanced salt solution. Two milliliters of RES-1 is added briskly over the entire surface. The dish may be set on a rocking platform if several samples are being processed simultaneously. The thick lysate is scraped to one side using the edge of a disposable plastic weigh boat and transferred to a 15-ml centrifuge tube. The dish is rinsed with 1 ml of RES-1 and this is combined with the first lysate. For cells grown in suspension culture, the washed cell pellet is first suspended in a small volume of balanced salt solution and then RES-1 is added quickly at the same RES-1-to-cell ratio. The lysate is sonicated at low power for 5 sec to shear the DNA and reduce its viscosity. Proteinase K (30 μΐ) is added and the mixture incu bated at 50° for 30 min. After cooling to room temperature, 0.2 ml sodium acetate and 7 ml cold 95% ethanol are added. The mixture is allowed to stand for 20 min at -20° for a precipitate of nucleic acids to form. After
40
I. ISOLATION, SYNTHESIS, AND DETECTION OF D N A AND R N A
centrifugation at 12,000 g for 10 min at 0°, the supernatant is removed and the precipitate is dissolved in 0.9 ml RES-1. The dissolved pellet of nucleic acids is transferred to a 1.5-ml polypropylene microcentrifuge tube. Proteinase K (10 /xl) is added and the sample is again incubated at 50° for 30 min. When cool, 0.1 ml of phenol/chloroform is added and the sample vortexed repeatedly over a 3-min period. The aqueous phase is recovered after centrifugation in a microcentrifuge for 5 min at room temperature. The organic layer is back extracted with 0.1 ml of RES-1 by vortexing for about 10 sec and centrifuging for 2 min. The combined aqueous fractions are then extracted once with 0.1 ml of chloroform to remove phenol by vortexing for 20 sec and centrifuging for 2 min. After this stage, to avoid contamination with exogenous ribonucleases it is necessary to employ precautions, such as wearing disposable gloves and using autoclaved reagents, tubes, and pipette tips. The aqueous phase is transferred to a 2-ml polypropylene microcentri fuge tube (Sarstedt, St. Laurent, Quebec, Canada). High molecular weight RNA is selectively precipitated with 7.5 μΐ 2.0 M acetic acid and 1 ml LiCl/ethanol. The sample is mixed thoroughly and held on ice for at least 4 hr (preferably 16 hr). Precipitated RNA is collected by centrifugation for 2 min; the supernatant (containing DNA and low molecular weight RNA) is carefully and completely removed using afinepipette tip, followed by brief centrifugation to remove any liquid left on the walls of the tube. The walls can be washed with 0.5 ml of a 1: 1 mixture of LiCl/ethanol and water; the solution should be added carefully, so as not to dislodge the pellet of RNA, and then removed by aspiration. The final pellet containing purified RNA is dissolved in CCS (0.4 ml). Note that LiCl-precipitated RNA is slow to dissolve. To lower the salt concentration further in preparation for electrophoresis, RNA is reprecipitated by the addition of 35 μΐ of sodium acetate and 1.0 ml of cold ethanol. The volume of CCS, sodium acetate, and ethanol should be reduced if the original cell number was low (<3 x 106 cells/10-cm dish) to reduce losses at the ethanol precipitation stage. After allowing the precipitate to form at - 20° for 20-30 min, the RNA is collected by centrifugation for 2 min. The RNA is reprecipitated once more from 0.2 ml of CCS with sodium acetate and ethanol. Depending on its intended use, the RNA may be dissolved in CCS or in sterile citrate/CDTA without SDS. The final preparation is stored frozen. Typical recovery of high molecular weight RNA is about 25 ^g/106 cultured cells. The content of RNA in leukocytes is much lower and the expected recovery is about 0.5 ^tg/lO6 cells. Note that CDTA absorbs significantly below 260 nm and so A263nm should be used to estimate the quantity of RNA recovered.
[3] EXTRACTION OF R N A AND D N A FROM MAMMALIAN CELLS
41
Denaturation of RNA prior to Northern Analysis Gel electrophoresis of "denatured" RNA samples can be carried out using 1.2% (w/v) agarose in 20 mM sodium phosphate, 2 mM CDTA, 1 M formaldehyde. RNA samples in CCS are denatured by mixing 10 μΐ RNA (3-10 μg), 5 μ,Ι formamide, and 5 μΐ FFP and heating at 55° for 30 min. Note that RNA will be degraded by this heating step if the pH is too low (
42
I. ISOLATION, SYNTHESIS, AND DETECTION OF D N A AND R N A
possible. The DNA is dissolved in 0.1 ml of CT containing 50 ^g/ml ribonuclease. It is shaken gently at first and then more vigorously as the solution becomes more viscous. When most is dissolved, the sample is incubated for 20 min at 37° to allow the ribonuclease to partially digest the RNA. The viscous solution is transferred to a 1.5-ml polypropylene tube and the original tube rinsed with 0.1 ml of DES. An equal volume of phenol/ chloroform is added and the sample extracted by vortexing repeatedly over a 5-min period or by shaking on a mechanical shaker for 20-30 min. After centrifugation for 2 min in a microcentrifuge, the aqueous layer and any interface material is transferred to a fresh tube. Fresh phenol/ chloroform is added and the extraction is repeated. The aqueous layer is recovered and clarified by a 5-min centrifugation, leaving behind any insoluble material. DNA is precipitated with 1 vol 95% ethanol (at room temperature) and the precipitate collected by centrifugation. The pellet is washed twice by suspending it in 1 ml ethanol with vortexing over several minutes to extract traces of phenol and SDS. Following brief centrifugation, the supernatant is discarded. Residual ethanol is removed under vacuum. The final pellet is dissolved in CT to give an estimated final concentra tion of about 500 μg DNA/ml. The solution should be very viscous (viscoelastic) and may take 24 hr or longer to dissolve completely. Store at 4°. Note that removal of precise volumes requires care because of the high viscosity of the solution. As for RNA, A263nm is used instead of A260nm t o estimate the quantity of DNA recovered. RNA contamination in DNA is conveniently estimated using a spectrofluorometer. About 1 ^g/ml DNA is dissolved in 0.5 ^g/ml ethidium bromide in CT. A fluorescence reading [excitation (Ex)520, emission (Em)590] is taken and then 10 ^cg/ml ribo nuclease is added. Fluorescence readings are taken again at 5-min intervals until readings are stable. A decrease in fluorescence indicates RNA is present; the percentage contamination can be estimated knowing that the specific fluorescence of ribosomal RNA is about one-half that of duplex DNA. Problems that May Be Encountered
Purification of DNA from cells that secrete large amounts of extracellu lar matrix material such as collagen, hyaluronic acid, or proteoglycans has presented problems, giving rise to material that coprecipitates with nucleic acids on alcohol precipitation. The same difficulty is not encountered during purification of RNA from the same cells using the procedure as described. In the case of DNA, extensive treatment of the first lysate with equal volumes of phenol/chloroform before alcohol precipitation and
[3] EXTRACTION OF RNA AND DNA FROM MAMMALIAN CELLS
43
proteinase K treatment may minimize the problem, but at a cost of a poorer yield of DNA. Concluding Remarks Extraction of RNA and of DNA from mammalian cells each present different problems. For RNA, the most serious potential difficulty is degra dation by endogenous ribonucleases. For DNA, poor yield and degrada tion by shearing can result if the concentration is too low. In the described procedure, potentially hazardous substances such as guanidinium thiocyanate, phenol, and chloroform are avoided or minimized. The general strat egy for both RNA and DNA purification is to treat with proteinase K at an early step so that alcohol precipitation can be used to concentrate the samples and allow further processing in microcentrifuge tubes. This decreases the volume of phenol needed and also ensures near-quantitative recovery of material. Both procedures give highly purified DNA or RNA in good yield from cultured cells and from blood cells. The applicability of the procedures to the extraction of nucleic acids from tissues and organs has not been established. Acknowledgments This work was supported by a grant from the Medical Research Council of Canada.
37
[3] Extraction of High Molecular Weight RNA and DNA from Cultured Mammalian Cells By H. C. BIRNBOIM
Introduction Although a large number of techniques have been described for the isolation of pure high molecular weight RNA and DNA from mammalian cells, no method is universally applicable because of different problems encountered with different cell and tissue types and different uses of the purified nucleic acids. In general, it is difficult to prepare both RNA and high molecular weight DNA from the same extract,1,2 because each presents separate difficulties. The most common problem encountered in the isolation of intact RNA is the presence of stable ribonucleases inside and outside of cells. Because RNA is single stranded, one cut by a ribonuclease may destroy its usefulness. Thus, effective ribonuclease inhibitors are needed from the moment of initial cell lysis. RNA is not shear sensitive but it is very alkali labile, so the pH must be controlled at the pH of maximal stability (6.5-7.0), particularly if it is to be heated. Other factors need to be considered to isolate high molecular weight DNA. Deoxyribonucleases are less of a problem because, compared to ribonucleases, they are generally more labile and inhibitable by chelators. Furthermore, DNA is a duplex molecule, so that cleavage of one of the two strands usually does not destroy its experimental usefulness. Its high viscosity makes it difficult to redissolve after alcohol precipita tion and it is sensitive to degradation by shearing. Preparation of intact, chromosome-size pieces of DNA requires special procedures such as embedding cells in agarose,3 which will not be considered here. The present chapter describes methods for the isolation of highly purified RNA and DNA in near-quantitative yield from small numbers of cultured mammalian cells and white blood cells. These nucleic acids are suitable for Southern and Northern transfers and for other applications. The RNA extraction procedure is slightly modified from one described earlier.4
1
F. D. Kury, C. Schneeberger, G. Sliutz, E. Kubista, H. Salzer, M. Medl, S. Leodolter, H. Swoboda, R. Zeillinger, and J. Spona, Oncogene 5, 1403 (1990). 2 J. Karlinsey, G. Stamatoyannopoulos, and T. Enver, Anal. Biochem. 180, 303 (1989). 3 D. C. Schwartz and C. R. Cantor, Cell {Cambridge, Mass.) 37, 67 (1984). 4 H. C. Birnboim, Nucleic Acids Res. 16, 1487 (1988). RECOMBINANT DNA METHODOLOGY II
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
38
I. ISOLATION, SYNTHESIS, AND DETECTION OF D N A AND R N A
Extraction of RNA The moment of initial disruption of a cell is the time that ribonucleases are released and can cause degradation of cellular RNA. Cell extraction should therefore be carried out in a solution containing strong denaturing agents and/or other inhibitors of ribonucleases. The solution should be at pH 6.5-7.0 (the pH of optimal stability for RNA). 5 Although guanidinium thiocyanate is useful as a denaturing agent and ribonuclease inhibitor, 6-10 a mixture of sodium dodecyl sulfate (SDS) and a low concentration of urea 11 is an effective inhibitor of potent leukocyte ribonucleases, which are more difficult to inhibit than pancreatic ribonuclease. 4 At the same time, this mixture effectively activates proteinase K.12 Once most of the protein is digested, nucleic acids can be concentrated by precipitation with alcohol. This strategy minimizes the volume of phenol required for subsequent extractions and improves recovery. DNA can be eliminated almost completely by precipitation of high molecular weight RNA with LiCl, obviating the need for ribonuclease-free deoxyribonuclease or buoy ant density sedimentation. Reagents For RNA Extraction and Denaturation RES-1: 0.5 M LiCl, 1 M urea, 1.0% SDS, 0.02 M sodium citrate, and 2.5 mMcyclohexanediaminetetraacetate (CDTA) (final pH 6.8). Filter before addition of SDS and store at room temperature Proteinase K: 5 mg/ml in water; store at - 2 0 ° Phenol/chloroform: 10 g phenol crystals (analytical grade) dissolved in 10 ml of chloroform. Store at room temperature, protected from light. The solution should be colorless. The use of recrystallized phenol is unnecessary Sodium acetate (pH 5.2) (2 M in sodium ion): For 100 ml of solution, 0.2 mol of sodium acetate is adjusted to pH 5.2 (as measured at 0.05 M) with acetic acid and made up to volume. Filter, autoclave, and store at room temperature over chloroform Acetic acid: 2.0 M 5
H. C. Birnboim, unpublished observation (1974). J. H. Chirgwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979). 7 G. Cathala, J. F. Savouret, B. Mendez, B. L. West, M. Karin, J. A. Martail, and J. D. Baxter, DNA 2, 329(1983). 8 S. A. Krawetz, J. C. States, and G. H. Dixon, J. Biochem. Biophys. Methods 12, 29 (1986). 9 P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987). 10 S. L. Berger and J. M. Chirgwin, this series, Vol. 180, p. 3. 11 R. Soeiro, M. H. Vaughan, and J. E. Darnell, J. Cell Biol. 36, 91 (1968). 12 H. Hilz, U. Wiegers, and P. Adamietz, Eur. J. Biochem. 56, 103 (1975). 6
[3] EXTRACTION OF R N A AND D N A FROM MAMMALIAN CELLS
39
LiCl/ethanol: Combine 3 vol of 5 M LiCl (which has been filtered and autoclaved) with 2 vol of 95% ethanol. Store at room temperature CCS: 1 mM sodium citrate, 1 mM CDTA, 0.1% SDS, adjusted to pH 6.8. Autoclave before addition of SDS. Filter and store at room tem perature FFP (formaldehyde/formamide/phosphate): FFP [100 μ,Ι 37% formalde hyde, 94 μ,Ι formamide, 6.6 μΐ 1 M sodium phosphate (pH 6.8)] is freshly prepared before use. RNA will be partially degraded during heating prior to Northern analysis if the pH is <6.0. Formamide tends to become acidic on standing and the highest grade of formamide available (e.g., Omnisolv grade from BDH Chemicals, Poole, En gland) should be used. It can be stored at 4° over mixed-bed resin. Formaldehyde may also be acidic. The pH of formamide, formalde hyde, or mixtures is conveniently monitored using 2 vol bromthymol blue (0.25 mg/ml in 0.5 mM NaOH) as a color indicator Where indicated, solutions are filtered through a 0.45-μ,πι membrane filter. Cyclohexanediamine tetraacetate is a chelator that, compared to ethylenediaminetetraacetate (EDTA), binds metal ions 100-1000 more strongly, is much more soluble in acid and alcohol, and is easier to prepare in concentrated solutions. It is available from Sigma Chemical Co. (St. Louis, MO). RNA Extraction Procedure for Cultured Cells Volumes given are for 3-5 x 106 cells grown as a monolayer in a 10-cm petri dish and can be readily scaled up or down according to the cell number and surface area. The dish is chilled on an aluminum plate in contact with ice. The culture medium is quickly removed by aspiration and the cell layer washed twice with cold balanced salt solution. Two milliliters of RES-1 is added briskly over the entire surface. The dish may be set on a rocking platform if several samples are being processed simultaneously. The thick lysate is scraped to one side using the edge of a disposable plastic weigh boat and transferred to a 15-ml centrifuge tube. The dish is rinsed with 1 ml of RES-1 and this is combined with the first lysate. For cells grown in suspension culture, the washed cell pellet is first suspended in a small volume of balanced salt solution and then RES-1 is added quickly at the same RES-1-to-cell ratio. The lysate is sonicated at low power for 5 sec to shear the DNA and reduce its viscosity. Proteinase K (30 μΐ) is added and the mixture incu bated at 50° for 30 min. After cooling to room temperature, 0.2 ml sodium acetate and 7 ml cold 95% ethanol are added. The mixture is allowed to stand for 20 min at -20° for a precipitate of nucleic acids to form. After
40
I. ISOLATION, SYNTHESIS, AND DETECTION OF D N A AND R N A
centrifugation at 12,000 g for 10 min at 0°, the supernatant is removed and the precipitate is dissolved in 0.9 ml RES-1. The dissolved pellet of nucleic acids is transferred to a 1.5-ml polypropylene microcentrifuge tube. Proteinase K (10 /xl) is added and the sample is again incubated at 50° for 30 min. When cool, 0.1 ml of phenol/chloroform is added and the sample vortexed repeatedly over a 3-min period. The aqueous phase is recovered after centrifugation in a microcentrifuge for 5 min at room temperature. The organic layer is back extracted with 0.1 ml of RES-1 by vortexing for about 10 sec and centrifuging for 2 min. The combined aqueous fractions are then extracted once with 0.1 ml of chloroform to remove phenol by vortexing for 20 sec and centrifuging for 2 min. After this stage, to avoid contamination with exogenous ribonucleases it is necessary to employ precautions, such as wearing disposable gloves and using autoclaved reagents, tubes, and pipette tips. The aqueous phase is transferred to a 2-ml polypropylene microcentri fuge tube (Sarstedt, St. Laurent, Quebec, Canada). High molecular weight RNA is selectively precipitated with 7.5 μΐ 2.0 M acetic acid and 1 ml LiCl/ethanol. The sample is mixed thoroughly and held on ice for at least 4 hr (preferably 16 hr). Precipitated RNA is collected by centrifugation for 2 min; the supernatant (containing DNA and low molecular weight RNA) is carefully and completely removed using afinepipette tip, followed by brief centrifugation to remove any liquid left on the walls of the tube. The walls can be washed with 0.5 ml of a 1: 1 mixture of LiCl/ethanol and water; the solution should be added carefully, so as not to dislodge the pellet of RNA, and then removed by aspiration. The final pellet containing purified RNA is dissolved in CCS (0.4 ml). Note that LiCl-precipitated RNA is slow to dissolve. To lower the salt concentration further in preparation for electrophoresis, RNA is reprecipitated by the addition of 35 μΐ of sodium acetate and 1.0 ml of cold ethanol. The volume of CCS, sodium acetate, and ethanol should be reduced if the original cell number was low (<3 x 106 cells/10-cm dish) to reduce losses at the ethanol precipitation stage. After allowing the precipitate to form at - 20° for 20-30 min, the RNA is collected by centrifugation for 2 min. The RNA is reprecipitated once more from 0.2 ml of CCS with sodium acetate and ethanol. Depending on its intended use, the RNA may be dissolved in CCS or in sterile citrate/CDTA without SDS. The final preparation is stored frozen. Typical recovery of high molecular weight RNA is about 25 ^g/106 cultured cells. The content of RNA in leukocytes is much lower and the expected recovery is about 0.5 ^tg/lO6 cells. Note that CDTA absorbs significantly below 260 nm and so A263nm should be used to estimate the quantity of RNA recovered.
[3] EXTRACTION OF R N A AND D N A FROM MAMMALIAN CELLS
41
Denaturation of RNA prior to Northern Analysis Gel electrophoresis of "denatured" RNA samples can be carried out using 1.2% (w/v) agarose in 20 mM sodium phosphate, 2 mM CDTA, 1 M formaldehyde. RNA samples in CCS are denatured by mixing 10 μΐ RNA (3-10 μg), 5 μ,Ι formamide, and 5 μΐ FFP and heating at 55° for 30 min. Note that RNA will be degraded by this heating step if the pH is too low (
42
I. ISOLATION, SYNTHESIS, AND DETECTION OF D N A AND R N A
possible. The DNA is dissolved in 0.1 ml of CT containing 50 ^g/ml ribonuclease. It is shaken gently at first and then more vigorously as the solution becomes more viscous. When most is dissolved, the sample is incubated for 20 min at 37° to allow the ribonuclease to partially digest the RNA. The viscous solution is transferred to a 1.5-ml polypropylene tube and the original tube rinsed with 0.1 ml of DES. An equal volume of phenol/ chloroform is added and the sample extracted by vortexing repeatedly over a 5-min period or by shaking on a mechanical shaker for 20-30 min. After centrifugation for 2 min in a microcentrifuge, the aqueous layer and any interface material is transferred to a fresh tube. Fresh phenol/ chloroform is added and the extraction is repeated. The aqueous layer is recovered and clarified by a 5-min centrifugation, leaving behind any insoluble material. DNA is precipitated with 1 vol 95% ethanol (at room temperature) and the precipitate collected by centrifugation. The pellet is washed twice by suspending it in 1 ml ethanol with vortexing over several minutes to extract traces of phenol and SDS. Following brief centrifugation, the supernatant is discarded. Residual ethanol is removed under vacuum. The final pellet is dissolved in CT to give an estimated final concentra tion of about 500 μg DNA/ml. The solution should be very viscous (viscoelastic) and may take 24 hr or longer to dissolve completely. Store at 4°. Note that removal of precise volumes requires care because of the high viscosity of the solution. As for RNA, A263nm is used instead of A260nm t o estimate the quantity of DNA recovered. RNA contamination in DNA is conveniently estimated using a spectrofluorometer. About 1 ^g/ml DNA is dissolved in 0.5 ^g/ml ethidium bromide in CT. A fluorescence reading [excitation (Ex)520, emission (Em)590] is taken and then 10 ^cg/ml ribo nuclease is added. Fluorescence readings are taken again at 5-min intervals until readings are stable. A decrease in fluorescence indicates RNA is present; the percentage contamination can be estimated knowing that the specific fluorescence of ribosomal RNA is about one-half that of duplex DNA. Problems that May Be Encountered
Purification of DNA from cells that secrete large amounts of extracellu lar matrix material such as collagen, hyaluronic acid, or proteoglycans has presented problems, giving rise to material that coprecipitates with nucleic acids on alcohol precipitation. The same difficulty is not encountered during purification of RNA from the same cells using the procedure as described. In the case of DNA, extensive treatment of the first lysate with equal volumes of phenol/chloroform before alcohol precipitation and
[3] EXTRACTION OF RNA AND DNA FROM MAMMALIAN CELLS
43
proteinase K treatment may minimize the problem, but at a cost of a poorer yield of DNA. Concluding Remarks Extraction of RNA and of DNA from mammalian cells each present different problems. For RNA, the most serious potential difficulty is degra dation by endogenous ribonucleases. For DNA, poor yield and degrada tion by shearing can result if the concentration is too low. In the described procedure, potentially hazardous substances such as guanidinium thiocyanate, phenol, and chloroform are avoided or minimized. The general strat egy for both RNA and DNA purification is to treat with proteinase K at an early step so that alcohol precipitation can be used to concentrate the samples and allow further processing in microcentrifuge tubes. This decreases the volume of phenol needed and also ensures near-quantitative recovery of material. Both procedures give highly purified DNA or RNA in good yield from cultured cells and from blood cells. The applicability of the procedures to the extraction of nucleic acids from tissues and organs has not been established. Acknowledgments This work was supported by a grant from the Medical Research Council of Canada.