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 procedure is gentle enough to allow the selective removal 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 processing of multiple samples at one time. 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, and then adding an appropriate volume of 10% SDS. The 10% SDS stock solution 1s pre- 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 1 OOg for 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,OOOg for 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,OOOg for 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 1 O7 cells 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 success of 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 1 O7 cells with Az6,jA2s0 values of approx 1.86 (31). These values compare favorably with those obtained using guanidinium thiocy- anate CsCl centnfugatron methods (8). 8 Merante et al. References 1. Graham, D E (1978) The isolatton of high molecular weight DNA from whole organisms of large ttssue masses. Anal Biochem 85,609-6 13. 2. Bowtell, D D. (1987) Rapid isolation of eukaryotic DNA Anal Bzochem 162, 463-465 3 Longmire, J L , Albright, K. L , Meincke, L. J , and Hildebrand, C E. (1987) A rapid and simple method for the isolation of high molecular weight cellular and chromosome-specific DNA m solutton without the use of organic solvents. Nuclezc Acids Res 15,859. 4 Owen, R. J. and Borman, P. (1987) A rapid biochemical method for punfymg htgh molecular weight bacterial chromosomal DNA for restriction enzyme analysts. Nuclex Acids Res. 15,363 1 5. Reymond, C. D. (1987) A rapid method for the preparation of multiple samples of eukaryotic DNA. Nuclezc Aczds Res. 15, 8 118 6. Miller, S A. and Polesky, H F (1988) A simple salting out procedure for extract- ing DNA from human nucleated cells Nuclezc Acids Res. 16, 12 15. 7. Grimberg, J , Nawoschik, S , Belluscio, L., McKee, R., Turck, A, and Etsenberg, A. (1989) A simple and efficient non-orgamc procedure for the isolation of genomtc DNA from blood Nuclerc Acids Res 17,839O. 8. Chtrgwm, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Isola- tion of biologically active ribonucleic acid from sources enriched m ribonuclease. Biochemzstry 18,5294-5299 9. Auffray, C and Rougeon, F. (1980) Puriflcatton of mouse rmmunoglobulm heavy- chain messenger RNAs from total myeloma tumour RNA. Eur J Bzochem 107, 303-3 14. 10. Elion, E A. and Warner, J. R. (1984) The major promoter element of rRNA tran- scription in yeast lies 2 Kb upstream Cell 39,663-673. 11 Chomczynski, P and Sacchi, N (1987) Single-step method of RNA extraction by acid guamdinium thiocyanate-phenol-chloroform extraction. Anal. Blochem. 162, 156-159. 12. Hatch, C L. and Bonner, W M. (1987) Direct analysis of RNA m whole cell and cytoplasmic extracts by gel electrophoresis. Anal. Blochem 162,283-290 13. Emmett, M. and Petrack, B. (1988) Rapid isolation of total RNA from mammalian tissues. Anal. Blochem. 174,658-661. 14. Gough, N M. (1988) Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. Anal Blochem 173,93-95. 15. Meter, R. (1988) A universal and efficient protocol for the tsolatton of RNA from tissues and cultured cells Nucleic Acrds Res. 16,234O. 16. Wilkinson, M. (1988) RNA isolation a mini-prep method. Nuclezc Aczds Res. 16, 10,933 17. Wilkinson, M (1988) A rapid and convenient method for isolation of nuclear, cyto- plasmic and total cellular RNA Nucleic Acids Res 16, 10,934. 18. Ferre, F and Garduno, F (1989) Preparation of crude cell extract suitable for amplification of RNA by the polymerase chain reaction. Nuclerc Acids Res 17,2 141 19. McEntee, C. M. and Hudson, A. P. (1989) Preparation of RNA from unsphero- plasted yeast cells (Saccharomyces cerevwae). Anal Blochem. 176,303-306 Isolation of RNA and DNA 9 20. Nemeth, G. G., Heydemann, A., and Bolander, M. E. (1989) Isolation and analysis of ribonucleic acids from skeletal tissues. Anal Blochem 183, 30 l-304 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 22. Schnntt, M E., Brown, T A., and Trumpower, B L. (1990) A rapid and simple method for preparatron of RNA from Saccharomyces cerevwae Nuclerc Acids Res l&3091 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 24. Krieg, P., Amtmann, E., and Sauer, G. (1983) The simultaneous extraction of high- molecular-weight DNA and of RNA from sohd tumours Anal Biochem 134, 288-294. 25. Mirkes, P E (1985) Simultaneous banding of rat embryo DNA, RNA and protein m cesium trifluroracetate gradients. Anal Blochem 148,37&383 26. Meese, E. and Blin, N. (1987) Simultaneous isolation of high molecular weight RNA and DNA from limited amounts of tissues and cells Gene Anal Tech. 4, 4549. 27. Zarlenga, D. S. and Gamble, H. R. (1987) Simultaneous isolatton of preparative amounts of RNA and DNA from Trichinella spirahsby cesium trrfluoroacetate isopycnic centrifugation. Anal. Blochem. 162,569-574 28. Chan, V T.-W , Fleming, K A , and McGee, J. 0 D (1988) Simultaneous extrac- tion from clinical biopsies of high-molecular-weight DNA and RNA: comparattve characterization by biotmylated and 32P-labeled probes on Southern and Northern blots. Anal Blochem 168, 16-24 29. Karlinsey, J., Stamatoyannopoulos, G., and Enver, T. (1989) Simultaneous purifi- cation of DNA and RNA from small numbers of eukaryotic cells. Anal Bzochem 180,303-306. 30. Coombs, L. M , Pigott, D , Proctor, A , Eydmann, M., Denner, J., and Knowles, M A. (1990) Simultaneous isolation of DNA, RNA and antrgenic protem exhibiting kinase activity from small tumour samples using guanidine tsothiocyanate. Anal. Bzochem. 188,338-343 3 1 Raha, S., Merante, F , Proteau, G , and Reed, J. K (1990) Simultaneous tsolation of total cellular RNA and DNA from tissue culture cells using phenol and hthmm chloride. Gene Anal Tech. 7, 173-177 32. Kirby, K. S. (1957) A new method for the isolatron of deoxyribonucleic acids: evidence on the nature of bonds between deoxyribonucleic acid and protein. Blochem J 66,495-504 33. Wallace, D. M. (1987) Large and small scale phenol extractions, m Methods ln Enzymology, vol 152 Guide to Molecular Clonmg Techniques (Berger, S. L. and Kimmel, A R., eds.), Academic, Orlando, FL, pp. 334 1 34. Sambrook, J , Fritsch, E F., and Maniatis, T. (1989) Molecular Clonzng. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring, Harbor, NY. 35. Davis, L G., Dibner, M. D., and Battey, J. F (1986) Basic Methods in MoZecuZur Brology Elsevter, New York. CHAPTER 2 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 bases will 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