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`US 20080003574Al
`
`c19) United States
`c12) Patent Application Publication
`Michalik et al.
`
`c10) Pub. No.: US 2008/0003574 Al
`Jan. 3, 2008
`(43) Pub. Date:
`
`(54) KITS FOR RNA EXTRACTION
`
`(22) Filed:
`
`Jun. 28, 2006
`
`(75)
`
`Inventors:
`
`Steve S. Michalik, St. Louis, MO
`(US); Scott A. Weber, St. Louis,
`MO (US); Carol A. Kreader, St.
`Louis, MO (US)
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`C12Q 1168
`C12P 19134
`
`(2006.01)
`(2006.01)
`
`Correspondence Address:
`SENNIGER POWERS (SGM)
`ONE METROPOLITAN
`SQUARE,
`FLOOR
`ST. LOUIS, MO 63102
`
`16TH
`
`(73) Assignee:
`
`Sigma-Aldrich Co., St. Louis, MO
`(US)
`
`(21) Appl. No.:
`
`11/476,269
`
`(52) U.S. Cl. ........................................... 435/6; 435/91.2
`
`(57)
`
`ABSTRACT
`
`The present invention provides methods and compositions
`for extracting RNA from cells. The cellular extract may be
`directly used in a variety of reactions, such as reverse
`transcription and PCR.
`
`Page 1
`
`Spectrum Ex. 1008
`IPR Petition - USP 10,000,795
`
`

`

`Patent Application Publication
`
`Jan. 3, 2008 Sheet 1 of 11
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`US 2008/0003574 Al
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`Patent Application Publication
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`Jan. 3, 2008 Sheet 2 of 11
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`FIG. 2A
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`Page 4
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`Page 5
`
`

`

`Patent Application Publication
`
`Jan. 3, 2008 Sheet 5 of 11
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`US 2008/0003574 Al
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`Page 6
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`Patent Application Publication
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`

`

`US 2008/0003574 Al
`
`Jan. 3, 2008
`
`1
`
`KITS FOR RNA EXTRACTION
`
`BACKGROUND OF THE INVENTION
`
`[0001] The present invention provides methods and com(cid:173)
`positions for extracting RNA from cells. Also provided are
`methods and compositions for performing reverse transcrip(cid:173)
`tion and PCR with these extracts.
`[0002] The ability to study nucleic acids in biological
`samples has been important in biological and biochemical
`research. Reverse transcription followed by the quantitative
`polymerase chain reaction (qRT-PCR) is one of the main
`methods used for measuring mRNA levels from a small
`number-of cells. RT-PCR is useful for detecting RNA spe(cid:173)
`cies such as in quantitative analysis of gene expression,
`validation of mRNA knockdown by siRNA, signal ampli(cid:173)
`fication in in-situ hybridizations, as well as for other appli(cid:173)
`cations.
`[0003] The application of RT-PCR and other methods in
`molecular biology require the extraction of RNA from
`biological samples. A number of approaches have been
`devised for performing such extractions. These approaches
`have included treating or manipulating cells in order to lyse
`the cells and release RNA, along with other cellular com(cid:173)
`ponents. Some techniques have lysed cells using enzymatic
`activity, for example, by treating the cells with an enzyme
`such as proteinase K. Other techniques have treated cells
`with chaotropes and/or detergents or have used freeze thaw(cid:173)
`ing or snap freeze techniques to lyse cells.
`[0004] One drawback of lysing cells using these tech(cid:173)
`niques is that the resulting crude lysate typically contains not
`only RNA, but also a large amount of other cellular com(cid:173)
`ponents. For example, most cells contain some type of
`RNase that may contribute to RNA degradation. High con(cid:173)
`centrations of RNase activity in the crude lysate may make
`it more difficult to maintain the integrity of the RNA in the
`lysate. Furthermore, the crude lysate may contain DNA,
`which may interfere with RT-PCR. Reagents added to lyse
`cells may also interfere with RT-PCR. Consequently, it is
`usually necessary to purify RNA from the crude lysate prior
`to use in RT-PCR reactions. RNA purification often includes
`organic extraction or silica binding, which require centrifu(cid:173)
`gation or vacuum filtration. In some instances, it may be
`necessary to treat the crude lysate with enzymes capable of
`degrading or inactivating contaminating cellular debris.
`[0005] Furthermore, many of the known techniques for
`extracting RNA from cells are labor intensive, often requir(cid:173)
`ing specialized equipment and/or numerous steps. For
`example, enzymatic lysis often requires a heating and/or
`incubation step to inactivate the enzymes prior to perform(cid:173)
`ing RT-PCR or other such reactions. Furthermore, tech(cid:173)
`niques such as freeze thawing or snap freezing may require
`specialized conditions and equipment in order to perform the
`lysis.
`
`SUMMARY OF THE INVENTION
`
`[0006] Among the various aspects of the invention is the
`provision of a method for extracting RNA from cells.
`Advantageously, the method does not require the use of
`enzymes or any specialized equipment such as a centrifuge,
`vacuum or pressure system, or freezing or heating devices.
`The cellular extract produced using the methods of the
`present invention may be directly used in reverse transcrip-
`
`tion or RT-PCR reactions without first purifying or isolating
`RNA from other cellular debris.
`[0007]
`In one aspect of the present invention is a kit
`comprising instructions for forming an extraction medium
`comprising about 0.1 % to about 10% by weight of a
`detergent and about 10 mM to about 5 M of a salt; reagents
`for forming the extraction medium, the reagents comprising
`a detergent selected from the group consisting of a non-ionic
`detergent, a zwitterionic detergent, and combinations
`thereof, and a salt selected from the group consisting of
`monovalent salts, divalent salts, and combinations thereof;
`and a reverse transcriptase.
`[0008] Other objects and features will be in part apparent
`and in part pointed out hereinafter.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0009] FIGS. lA and 1B show charts depicting the aver(cid:173)
`age Ct values for GAPDH (FIG. lA) or PGKl (FIG. 18)
`obtained in qRT-PCR with cellular extracts or purified RNA
`prepared with different extraction solutions or commercially
`available kits, as discussed in Example 1.
`[0010] FIGS. 2A, 2B, 2C, and 2D show charts depicting
`Ct values for GAPDH or PGKl with cellular extracts
`prepared using either Ambion (Austin, Tex.) Cells-to-Signal
`kit, or extraction solution B or E either with or without an
`RNase inhibitor, as discussed in Example 2. FIG. 2A depicts
`GAPDH Ct values for extracts prepared without RNase
`inhibitor; FIG. 2B depicts GAPDH Ct values for extracts
`prepared with RNase inhibitor; FIG. 2C depicts PGKl Ct
`values for extracts prepared without RNase inhibitor; and
`FIG. 2D depicts PGKl Ct values for extracts prepared with
`RNase inhibitor.
`[0011] FIG. 3 shows a chart depicting G6PD and LMNA
`Ct values for reaction mixtures in which 50 mM KC! was
`either added or omitted from the reaction mixture, as dis(cid:173)
`cussed in Example 4.
`[0012] FIG. 4 shows a chart depicting a plot of PGKl Ct
`values vs. number of Pane 1 cells per well for cellular
`extracts prepared using different amounts of an extraction
`solution, as discussed in Example 5.
`[0013] FIGS. SA and SB show charts depicting Ct values
`from one-step (FIG. SA) or two-step (FIG. SB) qRT-PCR
`performed using extracts of cells transfected with siRNA
`targeting a specific target ( designated "siRNA") or extracts
`of cells prepared using siControl (designated "non-target")
`and assays for the target mRNAs, as discussed in Example
`6. FIG. SC shows a chart comparing the percent knockdown
`for one-step and two-step RT-PCR for each siRNA target, as
`discussed in Example 6.
`[0014] FIGS. 6A and 6B show charts comparing the Ct
`values from two-step qRT-PCR performed using either
`probe-based (FIG. 6A) or SYBR-based (FIG. 6B) specific
`gene expression assays and RNA obtained using extraction
`solution E ( designated "X") or a commercially available kit
`( designated "Q"), as discussed in Example 7.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0015] The present invention is directed to methods, com(cid:173)
`positions, and kits for extracting RNA from cells. The
`
`Page 13
`
`

`

`US 2008/0003574 Al
`
`Jan. 3, 2008
`
`2
`
`cellular extract may be directly used in a variety of reactions,
`such as reverse transcription and PCR.
`
`Cell Populations
`
`[0016] The present invention provides methods and kits
`for extracting RNA from cell populations using an extraction
`medium that comprises a salt and a detergent. The cell
`populations may comprise any cell or virus that comprises
`RNA ( e.g., mRNA, tRNA, rRNA, or non-coding RNA). The
`cell may be any of a variety of different types of cells
`including, for example, eukaryotic cells such as fungal,
`protist, plant, or animal cells. Preferably the cell is a
`mammalian cell, such as a rodent, mouse, rat, hamster,
`primate, or human cell. The cells may be living, dead, or
`damaged, that is, having disruptions in the cell wall or cell
`membrane. The cell may be obtained from any source, as
`will be understood by those of skill in the art, including from
`a cell culture, from a sample collected from a subject (e.g.,
`from an animal including a human) or the environment, from
`a tissue sample or body fluid ( e.g., whole blood, plasma,
`serum, urine, or cerebral spinal fluid), and other such
`sources.
`[0017] The cell population may be directly contacted with
`the extraction medium, or alternately, the cell population
`may be first concentrated by methods such as centrifugation,
`binding to a surface through immunoadsorption or other
`interaction, or filtration, prior to contact with the extraction
`medium. Optionally, the number of cells in the population
`may be increased by growing the cells on culture plates or
`in a suitable liquid medium prior to concentration or direct
`extraction. Methods and media for growing cells are well
`known to those of skill in the art.
`[0018]
`In one exemplary embodiment, the cell population
`is prepared by growing the cells in a suitable medium,
`harvesting the cells, and optionally washing the cells to
`remove contaminants prior to contacting the cells with the
`extraction medium. For example, for cells in a cell suspen(cid:173)
`sion, the cells may be harvested from the growth medium by
`or by centrifugation at a force of from about 1 to about
`100,000xg, more preferably at a force of from about 100 to
`about 1,0000xg, and preferably about 300xg for about 0.01
`to about 1500 minutes, and preferably for about 5 minutes.
`The growth medium may be removed by any suitable
`method including, for example, aspiration. In one embodi(cid:173)
`ment, the cell pellet may be washed using a suitable wash
`solution (e.g., PBS), repelleted as described above, and the
`wash solution removed by aspiration. The resulting cell
`population may then be contacted with the extraction
`medium, as described herein. A similar method may be used
`to remove the growth media from cells attached to a sub(cid:173)
`strate ( e.g., a tube or plate). In this instance, the cells can be
`contacted directly with extraction media after removal of
`growth media without the need for harvesting cells via
`trypsinization and centrifugation.
`
`Extraction Medium
`
`[0019] Once a suitable cell population has been obtained,
`RNA may be extracted from the cells using an extraction
`medium. The extraction medium of the present invention
`causes the release of RNA from cells present in the sample.
`In one preferred embodiment, the extraction medium com(cid:173)
`prises a detergent, a salt, and optionally other components
`that aid in the extraction and/or in reverse transcription or
`
`PCR reactions. Without wishing to be bound by any par(cid:173)
`ticular theory, it is believed that the extraction medium
`ruptures the cells through the action of the detergent and the
`salt. The detergent aids in the extraction by perforating the
`cell membrane, while the salt renders the extraction medium
`hypertonic. Under these hypertonic conditions, RNA is
`released from the cytosol of the ruptured cells through
`osmotic pressure exerted on the cell wall and/or cell mem(cid:173)
`brane as the cell collapses in on itself. Advantageously,
`under these conditions it is believed the genomic DNA
`(gDNA) remains associated with the nucleus and other
`cellular debris. Likewise, fewer RNases are available to
`digest the extracted RNA because the RNases also remain
`trapped in the cellular debris, or in some instances, may be
`present in the extract only at insignificant concentrations.
`[0020] Typically, the detergents and their concentrations
`used in the extraction medium are selected so as not to
`interfere with any reactions in which the extract may be
`used, particularly reverse transcription or PCR. Preferably,
`the extraction medium comprises a non-ionic detergent
`and/or a zwitterionic detergent. Non-ionic detergents are
`particularly preferred for use in the extraction medium
`because unlike some other detergents commonly used to
`lyse cells, non-ionic detergents can perforate the cell mem(cid:173)
`brane to allow release of the RNA without rupturing the cell
`and/or organelles to such an extent that large amounts of
`DNA and other cellular components are also released.
`[0021] Examplary non-ionic detergents for use in the
`extraction medium include BigCHAP (i.e. N,N-bis[3-(D(cid:173)
`gluconamido )propyl]cholamide ); bis(polyethylene glycol
`bis[imidazoyl carbonyl]); polyoxyethylene alcohols, such as
`Brij® 30 (polyoxyethylene(4)lauryl ether), Brij® 35 (poly(cid:173)
`oxyethylene(23)lauryl ether), Brij® 35P, Brij® 52 (polyoxy(cid:173)
`ethylene 2 cetyl ether), Brij® 56 (polyoxyethylene 10 cetyl
`ether), Brij® 58 (polyoxyethylene 20 cetyl ether), Brij® 72
`(polyoxyethylene 2 stearyl ether), Brij® 76 (polyoxyethyl(cid:173)
`ene 10 stearyl ether), Brij® 78 (polyoxyethylene 20 stearyl
`ether), Brij® 78P, Brij® 92 (polyoxyethylene 2 oleyl ether);
`Brij® 92V (polyoxyethylene 2 oleyl ether), Brij® 96\7,
`Brij® 97 (polyoxyethylene 10 oleyl ether), Brij® 98 (poly(cid:173)
`oxyethylene(20)oleyl ether), Brij® 58P, and Brij® 700
`(polyoxyethylene(l00)stearyl ether); Cremophor® EL (i.e.
`polyoxyethylenglyceroltriricinoleat 35; polyoxyl 35 castor
`oil); decaethylene glycol monododecyl ether; decaethylene
`glycol mono hexadecyl ether; decaethylene glycol mono
`tridecyl ether; N-decanoyl-N-methylglucamine; n-decyl
`a-D-glucopyranoside; decyl ~-D-maltopyranoside; digito(cid:173)
`nin; n-dodecanoyl-N-methylglucamide; n-dodecyl a-D(cid:173)
`maltoside; n-dodecyl ~-D-maltoside; heptaethylene glycol
`monodecyl ether; heptaethylene glycol monododecyl ether;
`heptaethylene glycol monotetradecyl ether; n-hexadecyl
`~-D-maltoside; hexaethylene glycol monododecyl ether;
`hexaethylene glycol monohexadecyl ether; hexaethylene
`glycol monooctadecyl ether; hexaethylene glycol monotet(cid:173)
`radecyl ether; Igepal® CA-630 (i.e. nonylphenyl-polyethyl(cid:173)
`englykol, ( octylphenoxy )polyethoxyethanol, octylphenyl(cid:173)
`polyethylene glycol); methyl-6-O-(N-heptylcarbamoyl)-a(cid:173)
`D-glucopyranoside; nonaethylene glycol monododecyl
`ether; N-nonanoyl-N-methylglucamine; octaethylene glycol
`monodecyl ether; octaethylene glycol monododecyl ether;
`octaethylene glycol monohexadecyl ether; octaethylene gly(cid:173)
`col monooctadecyl ether; octaethylene glycol monotetrade(cid:173)
`cyl ether; octyl-~-D-glucopyranoside; pentaethylene glycol
`monodecyl ether; pentaethylene glycol monododecyl ether;
`
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`3
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`pentaethylene glycol monohexadecyl ether; pentaethylene
`glycol monohexyl ether; pentaethylene glycol monooctade(cid:173)
`cyl ether; pentaethylene glycol monooctyl ether; polyethyl(cid:173)
`ene glycol diglycidyl ether; polyethylene glycol ether W-1;
`polyoxyethylene 10 tridecyl ether; polyoxyethylene 100
`stearate; polyoxyethylene 20 isohexadecyl ether; polyoxy(cid:173)
`ethylene 20 oleyl ether; polyoxyethylene 40 stearate; poly(cid:173)
`oxyethylene 50 stearate; polyoxyethylene 8 stearate; poly(cid:173)
`oxyethylene bis(imidazolyl carbonyl); polyoxyethylene 25
`propylene glycol stearate; saponin from quillaja bark; sor(cid:173)
`bitan fatty acid esters, such as Span® 20 (sorbitan mono(cid:173)
`laurate), Span® 40 (sorbitane monopalmitate), Span® 60
`(sorbitane monostearate), Span® 65 (sorbitane tristearate),
`Span® 80 (sorbitane monooleate), and Span® 85 (sorbitane
`trioleate ); various alkyl ethers of polyethylene glycols, such
`as Tergitol® Type 15-S-12, Tergitol® Type 15-S-30, Tergi(cid:173)
`tol® Type 15-S-5, Tergitol® Type 15-S-7, Tergitol® Type
`15-S-9, Tergitol® Type NP-10 (nonylphenol ethoxylate),
`Tergitol® Type NP-4, Tergitol® Type NP-40, Tergitol®
`Type NP-7, Tergitol® Type NP-9 (nonylphenol polyethylene
`glycol ether), Tergitol® MIN FOAM lx, Tergitol® MIN
`FOAM 2x, Tergitol® Type TMN-10 (polyethylene glycol
`trimethylnonyl ether), Tergitol® Type TMN-6 (polyethylene
`glycol trimethylnonyl ether), Triton® 770, Triton® CF-10
`(benzyl-polyethylene glycol tert-octylphenyl ether), Triton®
`CF-21, Triton® CF-32, Triton® DF-12, Triton® DF-16,
`Triton® GR-5M, Triton® N-42, Triton® N-57, Triton®
`N-60, Triton® N-101 (i.e. polyethylene glycol nonylphenyl
`ether; polyoxyethylene branched nonylphenyl ether), Tri(cid:173)
`ton® QS-15, Triton® QS-44, Triton® RW-75 (i.e. polyeth(cid:173)
`ylene glycol 260 mono(hexadecyl/octadecyl) ether and
`1-octadecanol), Triton® SP-135, Triton® SP-190, Triton®
`W-30, Triton® X-15, Triton® X-45 (i.e. polyethylene glycol
`4-tert-octylphenyl ether; 4-(1,1,3,3-tetramethylbutyl)phe(cid:173)
`nyl-polyethylene glycol), Triton® X-100 (t-octylphenoxy(cid:173)
`polyethoxyethanol; polyethylene glycol tert-octylphenyl
`ether; 4-(1, 1,3,3-tetramethylbutyl)phenyl-polyethylene gly(cid:173)
`col), Triton® X-102, Triton® X-114 (polyethylene glycol
`tert-octy lpheny I ether;
`( 1, 1,3 ,3-tetramethylbutyl )pheny 1-
`polyethy lene glycol), Triton® X-165, Triton® X-305, Tri(cid:173)
`ton® X-405 (i.e. polyoxyethylene( 40)isooctylcyclohexyl
`ether; polyethylene glycol tert-octylphenyl ether), Triton®
`X-705-70, Triton® X-151, Triton® X-200, Triton® X-207,
`Triton® X-301, Triton® XL-SON, and Triton® XQS-20;
`tetradecyl-~-D-maltoside; tetraethylene glycol monodecyl
`ether; tetraethylene glycol monododecyl ether; tetraethylene
`glycol monotetradecyl ether; triethylene glycol monodecyl
`ether; triethylene glycol monododecyl ether; triethylene
`glycol monohexadecyl ether; triethylene glycol monooctyl
`ether; triethylene glycol monotetradecyl ether; polyoxyeth(cid:173)
`ylene sorbitan fatty acid esters, such as TWEEN® 20
`(polyethylene glycol sorbitan monolaurate), TWEEN® 20
`(polyoxyethylene (20) sorbitan monolaurate), TWEEN® 21
`(polyoxyethylene (4) sorbitan monolaurate), TWEEN® 40
`(polyoxyethylene (20) sorbitan monopalmitate), TWEEN®
`60 (polyethylene glycol sorbitan monostearate; polyoxyeth(cid:173)
`ylene (20) sorbitan monostearate), TWEEN® 61 (polyoxy(cid:173)
`ethylene (4) sorbitan monostearate), TWEEN® 65 ( poly(cid:173)
`oxyethylene
`(20)
`sorbitantristearate), TWEEN® 80
`(polyethylene glycol sorbitan monooleate; polyoxyethylene
`(20) sorbitan monooleate), TWEEN® 81 (polyoxyethylene
`(5) sorbitan monooleate), and TWEEN® 85 (polyoxyethyl(cid:173)
`ene (20) sorbitan trioleate); tyloxapol; n-undecyl ~-D-glu(cid:173)
`copyranoside, MEGA-8
`(octanoyl-N-methylglucamide);
`
`MEGA-9 (nonanoyl-N-methylglucamide); MEGA-10 (de(cid:173)
`canoyl-N-methy lglucamide); methy lhepty lcarbamoyl glu(cid:173)
`copyranoside; octy 1-glucopyranoside; octy 1-thioglucopyra(cid:173)
`noside;
`octyl-~-thioglucopyranoside;
`and
`var10us
`combinations thereof.
`In one embodiment, the non-ionic detergent is
`[0022]
`selected from the group consisting of alkyl glucosides, alkyl
`maltosides, alkyl thioglucosides, BigCHAP series deter(cid:173)
`gents, glucamides, polyoxyethylenes, and combinations
`thereof. Exemplary alkyl glucosides include n-decyl a-D(cid:173)
`glucopyranoside, methy 1-6-O-(N-hepty lcarbamoy I )-a-D(cid:173)
`glucopyranoside, n-undecyl ~-D-glucopyranoside, methyl(cid:173)
`heptylcarbamoyl glucopyranoside, octyl-glucopyranoside,
`and octyl-~-D-glucopyranoside. Exemplary alkyl malto(cid:173)
`sides include n-dodecyl a-D-maltoside, n-dodecyl ~-D(cid:173)
`maltoside, n-hexadecyl ~-D-maltoside, tetradecyl-~-D-mal(cid:173)
`toside, and decyl ~-D-maltopyranoside. Exemplary alkyl
`thioglucosides
`include octyl-~-thioglucopyranoside and
`octyl-thioglucopyranoside. Exemplary glucamides include
`n-dodecanoyl-N-methylglucamide, MEGA-8, MEGA-9,
`and MEGA-10. Exemplary polyoxyethylenes include poly(cid:173)
`oxyethylene alcohols, such as Brij® 30, Brij® 35, Brij®
`35P, Brij® 52, Brij® 56, Brij® 58, Brij® 72, Brij® 76,
`Brij® 78, Brij® 78P, Brij® 92; Brij® 92\7, Brij® 96\7, Brij®
`97, Brij® 98, Brij® 58P, and Brij® 700, Cremophor® EL,
`polyoxyethylene 10 tridecyl ether, polyoxyethylene 100
`stearate, polyoxyethylene 20 isohexadecyl ether, polyoxy(cid:173)
`ethylene 20 oleyl ether, polyoxyethylene 40 stearate, poly(cid:173)
`oxyethylene 50 stearate, polyoxyethylene 8 stearate, poly(cid:173)
`oxyethylene bis(imidazolyl carbonyl), polyoxyethylene 25
`propylene glycol stearate, Triton® 770, Triton® CF-10,
`Triton® CF-21, Triton® CF-32, Triton® DF-12, Triton®
`DF-16, Triton® GR-5M, Triton® N-42, Triton® N-57,
`Triton® N-60, Triton® N-101, Triton® QS-15, Triton®
`QS-44, Triton® RW-75, Triton® SP-135, Triton® SP-190,
`Triton® W-30, Triton® X-15, Triton® X-45, Triton®
`X-100, Triton® X-102, Triton® X-114, Triton® X-165,
`Triton® X-305, Triton® X-405, Triton® X-705-70, Triton®
`X-151, Triton® X-200, Triton® X-207, Triton® X-301,
`Triton® XL-SON, and Triton® XQS-20, and polyoxyethyl(cid:173)
`ene sorbitan fatty acid esters, such as TWEEN® 20,
`TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61,
`TWEEN® 65, TWEEN® 80, TWEEN® 81, and TWEEN®
`85. More preferably, the polyoxyethylene is Triton® X-100.
`In certain embodiments, the extraction medium
`[0023]
`may comprise a zwitterionic detergent. Preferably, the zwit(cid:173)
`terionic detergent is one that is compatible for use in reverse
`transcription and/or RT-PCR reactions. Examples of such
`zwitterionic detergents include CHAPS (i.e. 3-[(3-cholami(cid:173)
`dopropyl)dimethylammonio ]-1-propanesulfonate );
`CHAPSO (i.e. 3-[(3-cholamidopropyl)dimethylammonio]-
`2-hydroxy-l-propanesulfonate );
`N-dodecylmaltoside;
`a-dodecyl-maltoside; ~-dodecyl-maltoside; 3-( decyldim(cid:173)
`ethylammonio )propanesulfonate inner salt; 3-( dodecyldim(cid:173)
`ethylammonio )propanesulfonate inner salt; 3-(N,N-dimeth-
`ylmyristylammonio )propanesulfonate;
`3-(N,N-
`dimethyloctadecylammonio )propanesulfonate;
`3-(N,N-
`dimethyloctylammonio )propanesulfonate inner salt; 3-(N,
`N-dimethylpalmitylammonio )propanesulfonate;
`and
`betaines, including sulfobetaines and carbobetaines. Sulfo(cid:173)
`betaines include, for example, SB3-8, SB3-10, SB3-12,
`SB3-14, SB3-16, and SB3-18. Preferably, the zwitterionic
`detergent is a betaine.
`
`Page 15
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`Jan. 3, 2008
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`4
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`[0024] The extraction medium preferably comprises from
`about 0.1 % to about 10% by weight of the detergent, more
`preferably from about 0.5% to about 5% by weight of the
`detergent, and still more preferably about 1 % by weight of
`the detergent.
`In addition to the detergent, the extraction medium
`[0025]
`further comprises a salt. As will be recognized by those
`skilled in the art, certain salts may interfere with or inhibit
`reverse transcription or PCR reactions. As such, the salt and
`the concentration of salt used in the extraction medium is
`typically selected so as not to interfere with any of the
`reactions in which the extract may be used.
`[0026] Typically, the salt is either a monovalent salt, a
`divalent salt, or some combination thereof. Preferably, the
`salt is a monovalent salt. Exemplary monovalent salts
`include lithium fluoride (LiF), lithium chloride (LiCI),
`lithium bromide (LiBr), lithium iodide (Lil), sodium fluo(cid:173)
`ride (NaF), sodium chloride (NaCl), sodium bromide
`(NaBr), sodium iodide (Nal), potassium fluoride (KF),
`potassium chloride (KC!), potassium bromide (KBr), potas(cid:173)
`sium iodide (KI), rubidium fluoride (RbF), rubidium chlo(cid:173)
`ride (RbCI), rubidium bromide (RbBr), rubidium iodide
`(Rbl), cesium fluoride (CsF), cesium chloride (CsCI),
`cesium bromide (CsBr), and cesium iodide (Csl), among
`others. Preferably, the monvalent salt is selected from the
`group consisting of NaF, NaCl, NaBr, Nal, KF, KC!, KBr,
`KI, and combinations thereof. More preferably, the monova(cid:173)
`lent salt is NaCl or KC!.
`In certain embodiments, the extraction solution
`[0027]
`may comprise a divalent salt. Exemplary divalent salts
`include beryllium chloride (BeCl2 ), beryllium fluoride
`(BeF 2 ), beryllium bromide (BeBr2 ), beryllium iodide (Bel2 ),
`magnesium chloride (MgCl 2 ), magnesium fluoride (MgF 2 ),
`magnesium bromide (MgBr2 ), magnesium iodide (Mgl2 ),
`calcium chloride (CaCl 2 ), calcium fluoride (CaF2 ), calcium
`bromide (CaBr2 ), calcium iodide (Cal2 ), strontium chloride
`(SrCl2 ), strontium fluoride (SrF 2 ), strontium bromide
`(SrBr2 ), strontium iodide (Srl2 ), barium chloride (BaCl2 ),
`barium fluoride (BaF 2 ), barium bromide (BaBr2 ), and
`barium iodide (Bal2 ), among others. Preferably the divalent
`salt is selected from the group consisting of MgCl2 , MgF2 ,
`MgBr2 , Mgl2 . More preferably, the divalent salt is MgCl 2 .
`It will be recognized by those skilled in the art that
`[0028]
`some monovalent and/or divalent salts may have an inhibi(cid:173)
`tory effect on PCR. Therefore, in certain instances, it may be
`advantageous to add a chelating agent, as discussed below,
`to the cellular extract after RNA has been extracted and prior
`to performing PCR to control the amount of inhibitory
`monovalent and/or divalent salt in a PCR or RT-PCR reac(cid:173)
`tion mixture.
`[0029] As discussed above, the salt in the extraction
`medium acts to render the medium hypertonic, thus enabling
`RNA to be released from the ruptured cells by way of
`osmotic pressure exerted on the cell membrane or cell wall.
`It is thus preferable that the amount of salt present in the
`extraction medium be sufficient to render the medium hyper(cid:173)
`tonic (i.e., the concentration of salt in the extraction medium
`is higher than that in the cell). The concentration of salt in
`the extraction medium is preferably from about 150 mM to
`about SM, more preferably from about 200 mM to about 3.5
`M, and still more preferably is about 300 mM. If the
`extraction medium comprises a divalent salt, generally a
`lower amount of salt will be needed than if the extraction
`medium comprises a monovalent salt. For example, if the
`
`extraction medium comprises a divalent salt, the concentra(cid:173)
`tion of the salt in the extraction medium is preferably from
`about 10 mM to about 5 M, and more preferably is about 25
`mM to about 100 mM.
`[0030] Optionally, the extraction medium may also com(cid:173)
`prise certain agents to aid in stabilizing the extracted RNA.
`Typically, these agents and their concentrations are chosen
`so as not to interfere with reverse transcription, PCR, and/or
`other reactions in which the extract may be used. For
`example, in one embodiment, the extraction medium may
`comprise at least one RNase inhibitor. RNase inhibitors are
`known to those of skill in the art and include proteinacious
`RNase inhibitors such as human placental RNase inhibitors
`and porcine liver RNase inhibitors, anti-nuclease antibodies
`such as ANTI-RNase (Ambion, Inc., Austin, Tex.), clays
`such as macaloid and bentonite, polyanions, nucleotide
`analogs, reducing agents such as ~-mercaptoethanol, dithio(cid:173)
`threitol (DTT), dithioerythritol (DTE), and glutathione, and
`other such inhibitors. Preferably, the RNase inhibitor is a
`proteinacious RNase inhibitor. The RNase inhibitor may be
`present in the extraction medium in an amount of from about
`0.00001 units/µ! to about 1,000 units/µ!, and more preferably
`about 0.0001 units/µ! to about 1 unit/µ!. RNase inhibitors are
`particularly useful when the cell from which the RNA is to
`be extracted is located in a tissue, which may have more
`RNase activity than an individual cell or cell suspension.
`The RNase inhibitors may optionally be contained in the
`extraction medium and/or may be added to the cellular
`extract after extraction.
`[0031] Other agents that may be optionally included in the
`extraction medium or added to the extract after extraction
`include various molecules that selectively degrade DNA
`such as DNase I, Bal31 nuclease, T7 endonuclease, Neuro(cid:173)
`spora crassa nuclease, Lam