United States Patent (19)
`Heath
`
`54) LOW PH RNA ISOLATION REAGENTS,
`METHOD, AND KIT
`75 Inventor: Ellen M. Heath, Minnetonka, Minn.
`73 Assignee: Gentra Systems, Inc., Minneapolis,
`Minn.
`
`21 Appl. No.: 08/867,243
`22 Filed:
`Jun. 2, 1997
`Related U.S. Application Data
`62 Division of application No. 08/600,626, Feb. 13, 1996.
`(51) Int. Cl. ............................................... C07H 21/00
`52 U.S. Cl. .................... 536/25.4; 536/25.41; 536/23.1;
`435/91.3; 435/91.32; 436/177
`58 Field of Search ................................ 536/25.41, 25.4;
`435/91.3, 91.32; 436/177
`
`56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`4,745,057 5/1988 Beckage et al..
`4,843,155 6/1989 Chomczynski.
`5,010, 183 4/1991 Macfarlane.
`5,128.247 7/1992 Koller.
`5,393,672 2/1995 Ness et al..
`5,416.202 5/1995 Bernhard et al..
`5,496,562 3/1996 Burgoyne.
`FOREIGN PATENT DOCUMENTS
`O 554034 8/1993 European Pat. Off..
`WO/95284.09 10/1995 WIPO.
`
`OTHER PUBLICATIONS
`Berger & Kimmel “Guide to Molecular Cloning Tech
`niques” Methods in Enzymology vol. 152 (1987) Academic
`Press pp. 20–24, 33–38, 74, 215–241 and 492-495.
`Roitt et al"Immunology, Third Edition” Mosby Press (1993)
`pp. 1.5 and 12.2.
`Cooper “The Tools of Biochemistry” Wiley Intascience
`(1977) p. 358.
`Heth et al Am J of Human Genetics 57(4 Supp) 1995 A66.
`Sambrook et al Molecular Cloning: A Laboratory Manual,
`2nd ed, John Wiley and Sons, NY (1989) pp. 18.36–18.37
`and 6.55.
`Noonberg et al., “Effect of pH on RNA Degradation During
`Guanidinium Extraction,” BioTechniques, 19, 731-733
`(1995).
`
`USOO5973 137A
`Patent Number:
`11
`(45) Date of Patent:
`
`5,973,137
`Oct. 26, 1999
`
`Sambrook et al., Molecular Cloning. A Laboratory Manual,
`2nd Ed., 7.3–7.24, Cold Spring Harbor Press, Cold Spring
`Harbor, New York (1989).
`in Molecular Biology,
`Treco, Current
`Protocols
`13.12.1–13.12.3, John Wiley & Sons, New York (1989).
`Ullrich et al., “Rat Insulin Genes: Construction of Plasmids
`Containing the Coding Sequences,
`Science,
`196,
`1313–1319 (1977).
`Auffray et al., “Purification of Mouse Immunoglobulin
`Heavy-Chain Messenger RNAS from Total Myeloma Tumor
`RNA." Eur: J. Biochem., 107, 303-314 (1980).
`Ausubel et al., Current Protocols in Molecular Biology,
`4.0.3–4.5.2., John Wiley & Sons, New York (1989).
`Bugos et al., “RNAIsolation from Plant Tissues Recalcitrant
`to Extraction in Guanidine,” BioTechniques, 19, 734–737
`(1995).
`Chirgwin et al., “Isolation of Biologically Active Ribo
`nucleic Acid from Sources Enriched in Ribonuclease,” Bio
`chemistry, 18(24), 5294-5299 (1979).
`Chomczynski et al., “Single-Step Method of RNA Isolation
`by Acid Guanidinium Thiocyanate-Phenol-Chloroform
`Extraction.” Analytical Biochemistry, 162, 156-159 (1987).
`Cox, “The Use of Guuanidinium Chloride in the Isolation of
`Nucleic Acids.” Methods Enzymol., 12B, 120–129 (1968).
`Favaloro et al., “Transcription Maps of Polyoma Virus-Spe
`cific RNA: Analysis by Two-Dimensional Nuclease S1 Gel
`Mapping,” Methods Enzymol., 65, 718–749 (1980).
`Glisin et al., “Ribonucleic Acid Isolated by Cesium Chloride
`Centrifugation,” Biochem., 13, 2633–2637 (1974).
`Kohler et al., “Expression of bcr-abl Fusion Transcripts
`Following Bone Marrow Transplantation for Philadelphia
`Chromosome-Positive
`Leukemia,”
`Leukemia,
`4(8),
`541–547 (1990).
`Primary Examiner Anthony C. Caputa
`Assistant Examiner Heather A. Bakalyar
`Attorney, Agent, or Firm-Dorsey & Whitney LLP, Gregory
`J. Glover
`ABSTRACT
`57
`The present invention describes an RNA isolation process
`which utilizes low pH reagents. In addition, the reagents are
`leSS hazardous and are more Stable than those used in prior
`art methods. This rapid method may be used to obtain
`purified RNA from a variety of biological Sources including
`human whole blood, plant and animal tissues, cultured cells,
`body fluids, yeast, and bacteria.
`
`13 Claims, No Drawings
`
`Page 1
`
`Spectrum Ex. 1009
`IPR Petition - USP 10,000,795
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`

`

`1
`LOW PH RNA ISOLATION REAGENTS,
`METHOD, AND KIT
`This is a division of application Ser. No. 08/600,626,
`filed Feb. 13, 1996, which is incorporated herein by refer
`CCC.
`
`BACKGROUND OF THE INVENTION
`Ribonucleic acid (RNA) purified from biological material
`is utilized extensively for molecular biology research and is
`becoming an important tool in human clinical testing. Most
`commonly, the isolated RNA is characterized by size and
`quantity to provide diagnostic information about both nor
`mal and aberrant functioning of genes. For example, grOSS
`DNA rearrangements associated with common leukemias
`are detected by isolation and identification of abnormal,
`hybrid RNAs.
`Typically, there are three aspects of isolating Substantially
`undegraded RNA from biological samples: (1) the cells or
`viral protein coats are lysed to release RNA; (2) ribonu
`cleases (RNases) are inactivated to prevent RNA degrada
`tion; and (3) contaminants are removed to purify the prepa
`ration. Because of the abundance and stability of RNases in
`biological materials, it is important that cell or protein coat
`lysis and RNase inactivation be Substantially simultaneous.
`Therefore, in its simplest form, the isolation of RNA is
`reduced to just two main steps: (1) cell lysis (or protein
`denaturation)/RNase inactivation; and (2) RNA purification.
`Several lysing reagents have been formulated to lyse cells
`and/or viral protein coats and inactivate RNases Substan
`tially simultaneously. A lysate is created by mixing SuS
`pended cells (or biological fluid) with the lysing reagent, or
`by grinding tissues with a pestle in the presence of the lysing
`reagent, which facilitates penetration of the lysing reagent.
`The lysate reagent typically contains a detergent to dissolve
`cells and to Solubilize proteins and lipids. A Strong protein
`denaturant (i.e., denaturing agent) is usually added to aid in
`inactivating RNases. In addition, a strong reductant is often
`included to ensure complete protein denaturation.
`The most common detergents used in lysing reagent
`formulations are the anionic detergents Sodium dodecyl
`sulfate (SDS) and N-lauroyl sarcosine as described in
`Sambrook, et al., Molecular Cloning. A Laboratory Manual,
`2nd ed., 7.3–7.24, Cold Spring Harbor Press, Cold Spring
`Harbor, N.Y. (1989) and Ausubel, et al., Current Protocols
`in Molecular Biology, 4.0.4–4.5.3 and 13.12.1-13.12.3,
`John Wiley & Sons, New York (1989). Also, nonionic and
`cationic detergents have been described for this purpose by
`Favaloroet al., Methods Enzymol., 65, 718–749 (1980) and
`Macfarlane, (U.S. Pat. No. 5,010, 183), respectively.
`Typically, nonionic detergents are undesirable because they
`are generally ineffective at inactivating RNases in tissues
`with high nuclease activity. Cationic detergents are generally
`undesirable because they are more hazardous than nonionic
`and anionic detergents. For example, the rat intravenous
`LD50 is 1200 mg/kg for the nonionic detergent Triton X-100
`and 118 mg/kg for the anionic detergent SDS, but only 6.8
`mg/kg for the cationic detergent dodecyltrimethylammo
`nium bromide.
`Strong protein denaturants are commonly added to the
`lysing reagent to ensure inactivation of RNases. The most
`effective and widely used is guanidinium thiocyanate, which
`is described by Ullrich et al., Science, 196, 1313–1319
`(1977) and Chirgwin et al., Biochemistry, 19, 5294-5299
`(1979). Less commonly used as RNase inhibitors are
`organoclays, which are described by Ness et al. (U.S. Pat.
`No. 5,393,672).
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`Other denaturing agents that have been used are guanidine
`hydrochloride and urea, which are described by Cox in
`Methods Enzymol., 12, 120-129(1968) and Auffray et al.,
`Eur: J. Biochem., 107, 303-314(1980), respectively. These
`denaturing agents, however, are less effective at inactivating
`RNases than guanidinium thiocyanate. The addition of a
`proteolytic enzyme, Such as Proteinase K, to digest RNases
`is another Strategy used in RNA isolation techniques. This
`also is less effective than guanidinium thiocyanate because
`it is generally too slow at inactivating RNases causing RNA
`degradation, particularly in Solid tissue preparations.
`In addition to this primary denaturant, it is common
`practice to add a Second denaturant, Such as the Sulfhydryl
`reducing agent 2-mercaptoethanol, to the lysing reagent to
`ensure complete protein denaturation. This denaturant is
`highly toxic and has a pungent odor, and is therefore not
`easy to use. Furthermore, it is also Subject to oxidative
`degradation and therefore reduces the shelf-life of lysing
`reagents.
`An important factor to consider in the formulation of
`lysing reagents is pH. It has been shown by Noonberg et al.,
`BioTechniques, 19, 731-733 (1995) that for lysing reagents
`containing organic Solvents, the lower the pH, the lower the
`degree of RNA degradation, within the pH range of 5.5 to
`8.0. However, a review of RNA isolation methods indicates
`that the pH of the lysing reagent is no lower than 4.0
`(Chomczynski, U.S. Pat. No. 4,843,155), and can be as high
`as 9.0 (Bugos et al., BioTechniques 19, 734–737 (1995),
`with most in the neutral range of 7.0-8.0. Chomczynski
`teaches, however, that a pH of lower than 4 results in a
`Significantly lower degree of RNA isolation.
`After cell or protein coat lysis and RNase inactivation,
`RNA is purified by isolating it from the complex lysate.
`There are two general Strategies in widespread use for liquid
`phase purification of RNA. These are differential centrifu
`gation and Solvent extraction combined with Salt precipita
`tion.
`To separate RNA from deoxyribonucleic acid (DNA) and
`protein contaminants using differential centrifugation, typi
`cally the lysate is placed onto a Solution of cesium chloride
`as described by Glisin et al., Biochem., 13, 2633-2637
`(1974) and Chirgwin et al., Biochemistry, 19, 5294-5299
`(1979). Then the sample is centrifuged at high speed (at least
`130,000x g) for at least 12 hours to selectively sediment the
`RNA, leaving contaminants in the Supernatant fraction. This
`method has the disadvantages of being very time
`consuming, requiring the use of expensive ultracentrifuga
`tion equipment, and it does not efficiently recover low
`molecular weight RNAS, such as 5S ribosomal RNAS and
`transfer RNAS.
`The second strategy for RNA purification is to mix the
`lysate with both an organic Solvent (typically, phenol and
`chloroform) and a salt (typically, Sodium acetate). Phenol
`not only denatures proteins but, following centrifugation,
`causes the protein to collect at the interface between the
`organic and aqueous layers. Chloroform facilitates the Sepa
`ration of organic and aqueous phases. Such phenol-based
`reagents, however, are typically unstable during Storage due
`to oxidation.
`At low pH (e.g., 4-7), the addition of a high concentration
`(e.g., 2-3 molar) Salt Solution causes DNA to selectively
`precipitate So that following centrifugation, it too will col
`lect at the organic-acqueous interface. Thus, by combining
`the phenol eXtraction with Salt precipitation, both proteins
`and DNA collect at the interface following centrifugation,
`leaving RNA in the Supernatant. This is described, for
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`5,973,137
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`example, by Chomczynski et al., Anal. Biochem., 162
`156-159 (1987) and Chomczynski, EP 0554 034.
`The Salt Solutions generally used in Solvent extraction-Salt
`precipitation techniques are typically Sodium acetate Solu
`tions of pH 4.0 to pH 7.0 at concentrations of 2-3 molar. An
`alternative Salt, lithium chloride, Selectively precipitate
`RNA rather than the contaminating DNA. The addition of
`this Salt to the aqueous fraction, recovered after phenol
`chloroform extraction, is described by Ausubel et al., Cur
`rent Protocols in Molecular Biology, 4.0.4–4.5.3. John
`Wiley & Sons, New York (1989) and Auffray et al., Eur: J.
`Biochem., 107,303-314 (1980). However, a disadvantage of
`lithium chloride precipitation is that the low molecular
`weight RNAS are not recovered.
`Reagents required for isolating RNA in conventional
`methods are formulated typically using organic Solvents and
`other generally hazardous chemicals. For example, the raw
`materials in wide use are listed below, along with label
`precautions and toxicity information as obtained from Sigma
`Chemical Company. The toxicity data are given as LD50
`values where the lower the LD50 value, the more hazardous
`the compound. Generally, lysing and/or purification Solu
`tions contain: chloroform, which is highly toxic and may
`cause cancer, having an LD50 of 908 mg/kg (rat oral
`administration); guanidinium thiocyanate, which is consid
`ered harmful, having an LD50 of 300 mg/kg (mouse intra
`peritoneal injection), 2-mercaptoethanol, which is consid
`ered highly toxic and has a very Strong odor Stench, having
`an LD50 of 244 mg/kg (rat oral administration); and phenol,
`which is highly toxic, having an LD50 of 317 mg/kg (mouse
`oral administration).
`A method for DNA and RNA isolation that uses less
`hazardous compounds, Such as benzyl alcohol to replace
`phenol and chloroform, is disclosed by Ness et al., U.S. Pat.
`No. 5,393,672. Despite the lower toxicity of benzyl alcohol,
`it is still classified as harmful with an LD50 of 1230 mg/kg
`by rat oral administration. In addition, even leSS toxic
`organic Solvents require Special handling and disposal.
`Thus, there is a need in the field for a method that is less
`hazardous and/or does not involves the use of organic
`Solvents. In addition, there is a need for reagents that are
`more stable at room temperature (i.e., 20–30° C.). Also,
`there is a need for relatively rapid protocols to isolate RNA
`from a variety of biological materials, especially for routine
`45
`testing as found in clinical laboratories.
`SUMMARY OF THE INVENTION
`The present invention provides a kit for isolating RNA
`comprising instruction means for isolating Substantially
`50
`undegraded RNA from a biological Sample and a Cell Lysis
`Reagent. The Cell Lysis Reagent includes: an amount of an
`anionic detergent effective to lyse cells or protein coats
`sufficiently to release substantially undegraded RNA; a
`chelating agent, water, and an amount of a buffer effective
`to provide a pH of less than about 6 (preferably, less than
`about 5, and more preferably, less than about 4). The anionic
`detergent is preferably a dodecyl Sulfate Salt or N-lauroyl
`sarcosine. The chelating agent is preferably EDTA or CDTA.
`In addition to the Cell Lysis Reagent, the kit can include
`a Protein-DNA Precipitation Reagent comprising a sodium
`or potassium Salt in an amount effective to precipitate DNA
`and protein, water, and an amount of a buffer effective to
`provide a pH of less than about 6 (preferably, less than about
`5, and more preferably, less than about 4). Alternatively, the
`present invention provides a kit for isolating RNA compris
`ing instruction means for isolating Substantially undegraded
`
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`4
`RNA from a biological sample and a Protein-DNA Precipi
`tation Reagent comprising a Sodium or potassium Salt in an
`amount effective to precipitate DNA and protein, water, and
`an amount of a buffer effective to provide a pH of less than
`about 6. This reagent can be used in a method to isolate RNA
`from a biological Sample containing Substantially unde
`graded RNA released from cells or protein coats (i.e., a
`lysate) prepared using the Cell Lysis Reagent described
`above or a variety of known reagents for forming lysates.
`The kits of the present invention can also include an RNA
`Hydration Reagent comprising Substantially RNase-free
`deionized water for hydrating RNA once it is isolated from
`a biological Sample. For isolating RNA from mammalian
`whole blood, the kit can include an RBC Lysis Reagent
`comprising ammonium chloride, Sodium bicarbonate, and
`EDTA. For isolating RNA from yeast and Gram-positive
`bacteria, the kit can include a Cell Suspension Reagent
`comprising tris hydroxymethylaminomethane, EDTA, and
`Sorbitol; and a Lytic Enzyme Reagent comprising a lytic
`enzyme, glycerol, tris hydroxymethylaminomethane, and
`calcium chloride.
`The invention also provides a method for isolating RNA
`from a biological Sample. This method involves contacting
`the biological sample with the Cell Lysis Reagent described
`above to lyse cells or protein coats to form a lysate con
`taining substantially undegraded RNA. The substantially
`undegraded RNA is then separated from the lysate. This
`Separation Step preferably involves combining the lysate
`with the Protein-DNA Precipitation Reagent described
`above to precipitate DNA and protein. The substantially
`undegraded RNA is then Separated from the precipitated
`DNA and protein to form Substantially pure undegraded
`RNA.
`The present invention also provides a method for isolating
`RNA from mammalian blood comprising red and white
`blood cells. The method involves: contacting the blood with
`the RBC Lysis Reagent described above to lyse red blood
`cells and form a red cell lysate; Separating the white blood
`cells from the red cell lysate; contacting the white blood
`cells (and any cell-associated viruses) with the Cell Lysis
`Reagent described above to lyse the cells and protein coats
`to form a white cell lysate containing Substantially unde
`graded RNA, and Separating the Substantially undegraded
`RNA from the white cell lysate. This separating step pref
`erably involves: combining the white cell lysate with the
`Protein-DNA Precipitation Reagent described above to pre
`cipitate DNA and protein; and Separating the Substantially
`undegraded RNA from the precipitated DNA and protein to
`form substantially pure undegraded RNA.
`A further embodiment of the invention is a method for
`isolating RNA from a biological Sample, Such as yeast or
`Gram-positive bacteria. The method involves: combining
`the biological Sample with the Cell Suspension Reagent
`described above to form a cell Suspension; adding the Lytic
`Enzyme Reagent described above to the cell Suspension to
`form a mixture containing digested cells; Separating the
`digested cells from the mixture; contacting the digested cells
`with the Cell Lysis Reagent to lyse the cells and protein
`coats to form a cell lysate containing Substantially unde
`graded RNA, and Separating the Substantially undegraded
`RNA from the cell lysate. The separating step preferably
`includes combining the cell lysate with the Protein-DNA
`Precipitation Reagent to precipitate DNA and protein, and
`Separating the Substantially undegraded RNA from the pre
`cipitated DNA and protein to form substantially pure unde
`graded RNA.
`DETAILED DESCRIPTION OF THE
`INVENTION
`The present invention provides methods and kits that use
`aqueous reagents for isolating RNA from biological
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`Page 3
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`5,973,137
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`Samples. Such biological Samples include biological
`material, typically in an aqueous mixture, that contains
`RNA, including complex biological mixtures of procaryotic
`or eucaryotic cells. Typically, the biological material also
`includes DNA, proteins, and lipids. This includes, for
`example, biological fluids Such as blood, Saliva, and cere
`broSpinal fluid, Solid animal tissueS Such as heart, liver, and
`brain, animal waste products Such as feces and urine, plant
`tissues, yeasts, bacteria, Viruses, mycoplasmas, fungi,
`protozoa, rickettsia, and other Small microbial cells.
`Preferably, the methods and kits of the present invention
`provide Substantially undegraded RNA. AS used herein,
`“undegraded RNA means nondigested or intact RNA,
`which can be readily determined by one of skill in the art
`using Standard techniques. That is, the RNA is not damaged
`by enzymatic or chemical means during the isolation meth
`ods of the present invention. Preferably, the methods and kits
`of the present invention isolate a wide range of RNAS, Such
`as ribosomal RNA, messenger RNA, transfer RNA, and
`viral RNA, all of which can be recovered over a wide
`molecular weight range.
`Using these methods and kits, RNA of Substantially high
`yield can be obtained that is at least comparable to that
`obtained using conventional methods. Preferably, the iso
`lated RNA is substantially pure, which can be determined by
`the absence of Significant amounts of contaminating Sub
`stances Such as DNA and proteins, that could interfere with
`Subsequent analyses, Such as the Sensitive assay reverse
`transcriptase-polymerase chain reaction (RT-PCR). Thus,
`the isolated RNA is Suitable for use in Subsequent analyses
`known to those of skill in the art.
`The process consists of cell or protein coat lysis and
`RNase inactivation by combining the biological material
`with a lysing reagent containing an anionic detergent at low
`pH to form a lysate. As used herein, “lysis” refers to the
`destruction of a cell by rupture of its membranes or envelope
`as well as the denaturation of a viral protein coat. This is
`followed by RNA purification using a high concentration,
`low pH Salt reagent to Selectively remove contaminating
`40
`DNA and proteins. The final steps use common methods
`known to those of skill in the art. These steps are: (1) RNA
`concentration using standard precipitation methods; and (2)
`RNA hydration using a Standard hydration Solution, Such as
`RNase free water.
`The reagents used in the methods and kits of the present
`invention contain generally leSS hazardous components than
`many conventional RNA isolation reagents. Although, lower
`alcohols (i.e., (C-C)alkanols) may be used in concentrat
`ing RNA and/or removing residual Salt, the aqueous reagents
`of the present invention are Substantially free of organic
`Solvents. AS used herein, “Substantially free” means leSS
`than about 1%, and typically less than 0.6%, volume/
`Volume. Furthermore, they are at a lower pH than many
`conventional RNA isolation reagents. In addition, the
`reagents described in the present invention are much more
`Stable than conventional RNA isolation reagents. Generally,
`the aqueous reagents of the present invention consist of
`aqueous formulations of common Salts and detergents that
`are stable for at least about 18 months at room temperature
`(20–30° C).
`The first reagent, referred to herein as a “Cell Lysis
`Reagent, includes an anionic detergent dissolved in water.
`The reagent is buffered to a pH of less than about 6,
`preferably, less than about 5, and more preferably, less than
`about 4. The pH of all reagents described herein can be
`determined using a Standard laboratory pH meter with no
`
`6
`Specific Sample preparation. This reagent lyses cells and
`protein coats (e.g., as for viral RNA), and inactivates
`RNases rapidly enough that RNA is released from the cells
`or protein coats Substantially undegraded, to form a lysate.
`Preferably, the pH of this reagent is at least about 2 and more
`preferably, at least about 3.
`Suitable anionic detergents are those that are Soluble in
`water at a level of at least about 0.5% weight/volume, based
`on the total Volume of the reagent, and are capable of lysing
`cells and/or Solubilizing proteins and lipids at this concen
`tration. Such anionic detergents include, but are not limited
`to, salts (e.g., Sodium, potassium, and lithium salts) of
`dodecyl sulfate as well as N-lauroyl sarcosine. Preferably,
`the anionic detergent is a dodecyl Sulfate Salt. The anionic
`detergent is present in an amount effective to lyse cells and
`denature proteins causing the release of Substantially unde
`graded RNA. Preferably, it is present in an amount of about
`0.5-3%, more preferably, about 1-2.5%, and most prefer
`ably about, 1.8-2.2% weight/volume, based on the total
`Volume of the reagent.
`The pH of the Cell Lysis Reagent is maintained at less
`than about 6 using a buffer, Such as a citrate buffer, although
`a citrate buffer is not a requirement as long as the buffer is
`capable of providing a pH of less than about 6 in aqueous
`media. For example, bufferS Such as acetate, glycine,
`phthalate, aconitate, and Succinate can be used. Preferably,
`this pH is maintained using Sodium citrate and citric acid in
`combination. Preferably, the molar ratio of sodium citrate to
`citric acid is about 1:0.2 to about 1:13, and more preferably,
`about 1:2 for a pH of less than about 4. Preferably, a pH of
`less than about 6 is maintained using Sodium citrate at about
`10-100 mM, more preferably, 50-90 mM, and most
`preferably, 66-70 mM, concentration, based on the total
`volume of the reagent. Preferably, this pH is maintained
`using citric acid at 80-160 mM, more preferably, about
`100–150 mM, and most preferably, about 130–134 mM,
`concentration, based on the total Volume of the reagent.
`In addition to the anionic detergent and buffer, this first
`reagent includes a chelating agent. Suitable chelating agents
`are those capable of chelating divalent cations in aqueous
`media. Such chelating agents include, but are not limited to,
`ethylene diamine tetraacetate (EDTA) and cyclohexane
`diamine tetraacetate (CDTA). Preferably, the chelating agent
`is EDTA. A chelating agent is used in an amount effective to
`reduce DNase activity so that DNA is preferably released
`from the cells Substantially undegraded to facilitate its
`Subsequent removal. Preferably, the chelating agent is
`present in an amount of about 0.1-100 mM, more preferably
`about 1-20 mM, and most preferably about 8-12 mM, based
`on the total Volume of the reagent.
`The second reagent, referred to herein as a “Protein-DNA
`Precipitation Reagent, includes a Sodium or potassium Salt
`dissolved in water. The reagent is buffered to a pH of less
`than about 6, preferably, less than about 5, and more
`preferably, less than about 4. It is used for purification of the
`RNA. It includes a relatively high salt concentration, which
`causes contaminants Such as DNA and protein to be Selec
`tively precipitated, thereby enabling them to be removed by
`centrifugation. Because of the relatively low pH, the RNA
`remains in solution. Preferably, the pH of the Protein-DNA
`Precipitation Reagent is at least about 2, and more
`preferably, at least about 2.5.
`Suitable Salts for use in this Second reagent are those that
`are Soluble in water and are capable of causing precipitation
`of DNA and proteins from a lysate. Such salts include, but
`are not limited to, Sodium Salts. Such as Sodium chloride and
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`Sodium acetate, potassium Salts Such as potassium chloride
`and potassium acetate. Preferably, the Salt is Sodium chlo
`ride. The salt is present in this Protein-DNA Precipitation
`Reagent in an amount effective to precipitate a Sufficient
`amount of DNA and proteins out of a sample such that they
`do not interfere in the subsequent analysis of RNA.
`Preferably, the Salt is present at a concentration of about
`2-5.5 M, more preferably, at about 3–4.5 M, and most
`preferably, at about 3.8-4.2 M, based on the total volume of
`the reagent.
`The pH of the Protein-DNA Precipitation Reagent is
`maintained at less than about 6 using a buffer, Such as a
`citrate buffer, although a citrate buffer is not a requirement
`as long as the buffer is capable of providing a pH of less than
`about 6 in aqueous media. For example, bufferS Such as
`acetate, glycine, phthalate, aconitate, and Succinate can be
`used. Preferably, this pH is maintained using Sodium citrate
`and citric acid in combination. Preferably, the molar ratio of
`Sodium citrate to citric acid is about 1:0.2 to about 1:13, and
`more preferably, about 1:2 for a pH of less than about 4.
`Preferably, a pH of less than about 6 is maintained using
`Sodium citrate at about 1-30 mM, more preferably, 10-25
`mM, and most preferably, 15-20 mM, concentration.
`Preferably, this pH is maintained using citric acid at 10-60
`mM, more preferably, about 20-50 mM, and most
`preferably, about 30-35 mM, concentration.
`Both the Cell Lysis Reagent and the Protein-DNA Pre
`cipitation Reagent include water and Substantially no
`organic Solvents or other similarly hazardous components.
`Preferably, the water is deionized. More preferably, the
`water is deionized to a level Such that its resistance is greater
`than about 17 megohms. Most preferably, the water is
`deionized to this level of purity and further purified by
`filtering through a 0.2 uM pore size filter.
`Another aspect of this invention involves the combination
`of the two reagents, Cell Lysis Reagent and Protein-DNA
`Precipitation Reagent, with one or more optional, ancillary
`reagents. These ancillary reagents include reagents known to
`one of skill in the art for nucleic acid purification. The
`methods and kits of the present invention, however, are not
`limited to the use of these specific ancillary reagents, as one
`of skill in the art may use other reagents and/or techniques
`to achieve the same purpose. Also, each of the Cell Lysis
`Reagent and the Protein-DNA Precipitation Reagent can be
`used with other reagents and/or techniques if desired.
`A first ancillary reagent is Substantially RNase-free deion
`ized water, which is used to hydrate the purified RNA at the
`final Step in the RNA isolation process. This reagent is
`referred to herein as “RNA Hydration Reagent.” It includes
`water deionized to a level Such that its resistance is greater
`than about 17 megohms and further purified by filtering
`through a 0.2 uM pore size filter. In addition, the deionized
`water is treated with diethylpyrocarbonate (DEPC), or simi
`lar such material, to inactivate RNases. Preferably, DEPC is
`initially present in the deionized water at a concentration of
`about 0.05–0.1%, and more preferably 0.06–0.07%
`(volume/volume), based on the total volume of the water.
`The DEPC is mixed with the deionized water and held at
`room temperature for about 3–24 hours. Then the solution is
`heated under conditions effective to Substantially completely
`decompose the DEPC to CO and ethanol. This typically
`occurs at a temperature of at least about 100° C. and a
`pressure of at least about 20 psi (pounds per Square inch) for
`at least about 40 minutes in a Standard autoclave. Thus,
`when ready for use, the RNA Hydration Reagent is substan
`tially free of organic components (i.e., less than about 1%,
`and typically less than about 0.6% volume/volume).
`
`15
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`25
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`35
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`40
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`45
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`50
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`55
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`60
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`65
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`5,973,137
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`8
`The Second ancillary reagent is a red blood cell lysing
`reagent used to lyse red blood cells and facilitate Subsequent
`isolation of RNA from the white blood cells contained in
`mammalian whole blood. This reagent is referred to herein
`as the “RBC Lysis Reagent” and contains ammonium
`chloride, sodium bicarbonate, and EDTA. Preferably, the
`ammonium chloride is present in the RBC Lysis Reagent at
`a concentration of about 140-150 mM, more preferably, at
`about 142-146 mM, based on the total volume of the
`reagent. Preferably, the Sodium bicarbonate is present at a
`concentration of about 0.5-5 mM, and more preferably, at
`about 0.5-2 mM, based on the total volume of the reagent.
`Preferably, the EDTA is present at a concentration of about
`0.5-10 mM, and more preferably, at about 0.75–1.25 mM,
`based on the total volume of the reagent. RBC Lysis Reagent
`contains deionized water, preferably deionized to the level
`of purity described above, and further purified by filtration
`using a filter of about 0.2 uM pore size.
`When combined with mammalian whole blood, the RBC
`Lysis Reagent forms a red cell lysate containing Substan
`tially intact white blood cells. It can also contain viruses
`with Substantially intact protein coats. The white blood cells
`(and any cell-associated viruses that may be present) are
`then separated from the red cell lysate. The Cell Lysis
`Reagent is then combined with the white blood cells to lyse
`the white cells and protein coats (of the cell-associated
`viruses) to form a white cell lysate.
`The third and fourth ancillary reagents are used together
`to isolate RNA from yeast and Gram-positive bacteria. The
`reagents are referred to herein as "Cell Suspension Reagent'
`and “Lytic Enzyme Reagent.” They are used in the first steps
`of the RNA isolation procedure to digest cell walls as
`described in Example 6 below. The Cell Suspension Reagent
`is combined with a biological Sample to form a cell Suspen
`Sion. The Lytic Enzyme Reagent is combined with the cell
`Suspension to form a mixture containing digested cells.
`These digested cells are then Separated from the mixture by
`centrifugation, for example, and then contacted with the Cell
`Lysis Reagent.
`The Cell Suspension Reagent preferably has a pH of
`about 7-8.5, and