I 1111111111111111 1111111111 11111 11111 11111 111111111111111 lll111111111111111
`USO 10000795B2
`
`c12) United States Patent
`Birnboim et al.
`
`US 10,000,795 B2
`(IO) Patent No.:
`Jun.19,2018
`(45) Date of Patent:
`
`(54) STABILIZING COMPOSITIONS AND
`METHODS FOR EXTRACTION OF
`RIBONUCLEIC ACID
`
`(71) Applicant: DNA Genotek Inc., Kanata (CA)
`
`(72)
`
`Inventors: Hyman Chaim Birnboim, Ottawa
`(CA); Adele Jackson, Stittsville (CA)
`
`(73) Assignee: DNA GENOTEK INC., Ottawa (CA)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days. days.
`
`(21) Appl. No.: 15/160,712
`
`(22) Filed:
`
`May 20, 2016
`
`(65)
`
`Prior Publication Data
`
`8,158,357 B2
`8,470,536 B2
`2002/0081575 Al
`2002/0146677 Al
`2003/0170694 Al
`2005/0019814 Al
`2006/0275801 Al
`2007 /0087369 Al
`2009/0162866 Al
`2009/0162924 Al
`2010/0099149 Al
`2010/0273218 Al
`2015/0104803 Al
`
`4/2012 Birnboim et al.
`6/2013 Birnboim et al.
`6/2002 Small et al.
`10/2002 Augello et al.
`9/2003 Wall et al.
`1/2005 Laugharn et al.
`12/2006 Henkin
`4/2007 Chen et al.
`6/2009 Birnboim
`6/2009 Birnboim
`4/2010 Birnboim et al.
`10/2010 Birnboim et al.
`4/2015 Birnboim
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`WO
`WO
`WO
`WO
`WO
`
`10219117 Cl
`1999/000521 Al
`2003/104251 A2
`2004/033470 A2
`2007 /068094 Al
`2007/109586 A2
`
`10/2003
`1/1999
`12/2003
`4/2004
`6/2007
`9/2007
`
`US 2016/0340713 Al
`
`Nov. 24, 2016
`
`OTHER PUBLICATIONS
`
`(62)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Related U.S. Application Data
`
`Division of application No. 12/444,447, filed as
`application No. PCT/CA2007/001785 on Oct. 5,
`2007, now abandoned.
`
`Provisional application No. 60/949,778, filed on Jul.
`13, 2007, provisional application No. 60/866,985,
`filed on Nov. 22, 2006, provisional application No.
`60/828,563, filed on Oct. 6, 2006.
`
`Int. Cl.
`C12Q 1168
`C12N 15110
`U.S. Cl.
`CPC ....... C12Q 116806 (2013.01); C12N 1511003
`(2013.01)
`
`(2018.01)
`(2006.01)
`
`Field of Classification Search
`None
`See application file for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
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`(Continued)
`
`Primary Examiner - Robert M Kelly
`(74) Attorney, Agent, or Firm - Lathrop Gage LLP;
`James H. Velema, Esq.
`
`(57)
`
`ABSTRACT
`The present invention provides a composition and method
`for stabilizing ribonucleic acid (RNA) from biological
`samples such that the ribonucleic acid within the sample
`remains stable at room temperature. The composition com(cid:173)
`prises an anionic detergent and a buffering agent at a pH of
`about 5 to about 8.2 and is used in methods for extracting
`and storing ribonucleic acid from the biological sample.
`
`18 Claims, 17 Drawing Sheets
`
`Page 1
`
`Spectrum Ex. 1001
`IPR Petition - USP 10,000,795
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`

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`US 10,000,795 B2
`Page 2
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`(56)
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`Do NOT eat,drink, smnke or chew gum for 30 mh"l1.u:«s before glvln~¥_?1.n saliva sam I~
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`Step 5
`St~p 1
`Put fl $~"t\.;:.H a~no~nt
`Continut:: sµ1ttln9
`Shake the r.losed
`.A:. soc~n :;:i~ you f1n~$h
`De NOT swallow.
`<>I' tablr; wg:ar rn thfs
`(Of'lt-aine1 qukkly for
`spltting,caretuHy •~hp
`unttl the volumi;- of
`f,5 saliva gijthers in
`yaur rnr;:,uih, ;µ!t B,
`the container and
`for 1 O srzoo~-d~ to ~n!x
`,.,Uva fnot hc!udino
`palrn ofy<'ur hmw
`the ft">arn) fei.?Lht~§,~
`you, ,,.Uva wlth the
`;_md th•w toudi the
`into tht: <XH~tainer.
`t,ghtrm i'irmi:,,·. A !!qukf
`wrn b~ reteaseci fn)rn
`ifl,, 'ft,! to' !~",fii, .is
`top of yqvr ton,;_we
`Hquid fnJrn the cap.
`~hown ln pktore#:3.
`to the sugar. The
`tht~ CI~P~
`sugar;,vHt h~p Y<)U
`mak~ :S'1Hv~. J<€~p
`the ~aHvti 911<l .-:.:ug~~
`in yo~1r mouth for JU ..
`15 ~ei::onds withc•\.lt
`L sw,1!iowing.
`
`·----~l ______ J
`
`!
`
`Fig~ 15
`
`Page 20
`
`

`

`US 10,000,795 B2
`
`1
`STABILIZING COMPOSITIONS AND
`METHODS FOR EXTRACTION OF
`RIBONUCLEIC ACID
`
`RELATED APPLICATIONS
`
`This application is a division of U.S. patent application
`Ser. No. 12/444,447, filed Oct. 22, 2009, now abandoned,
`which is a 35 U.S.C. § 371 filing of International Patent
`Application No. PCT/CA2007/001785, filed Oct. 5, 2007,
`which claims priority to U.S. Provisional Patent Application
`Nos. 60/828,563; 60/866,985, and 60/949,778 the contents
`all of which are hereby incorporated by reference in their
`entirety.
`
`FIELD OF THE INVENTION
`
`The field of the invention generally relates to composi(cid:173)
`tions and methods for storage and/or isolation of ribonucleic
`acids from bodily fluid(s), and/or secretion(s), (e.g., saliva,
`mucous), and/or tissue(s).
`
`BACKGROUND
`
`The importance of detection and analysis of ribonucleic
`acid (RNA) is becoming increasingly evident. For example,
`a large number of pathogenic mammalian viruses ( e.g.
`SARS-CoA, Influenza virus, Measles virus, Rabies virus,
`Dengue fever virus, Respiratory Syncytial Virus (RSV),
`HIV and Hepatitis A, C-E virus) have genomes based on
`RNA rather than DNA. Detection and/or analysis of such
`RNA are potentially of great importance, yet an accepted
`method that is optimal for collecting, preserving/stabilizing,
`transporting and extracting RNA has not yet been devel(cid:173)
`oped.
`RNA is a labile compound and the widespread adoption
`for routine use of RNA as an analyte in detection and
`analysis of RNA has been limited because of its labile
`nature. The sugar-phosphate backbone of RNA is particu- 40
`larly sensitive to breakdown ( degradation, hydrolysis) by
`alkaline solutions. It is also sensitive to breakdown by acidic
`solutions. The pH of maximum stability of RNA is generally
`assumed to be about neutral, but this has not previously been
`determined precisely.
`RNA can also be degraded enzymatically by endoribo(cid:173)
`nucleases ( e.g., pancreatic ribonuclease ). Ribonuclease
`activity has previously been identified in human saliva
`(Bardon and Shugar, 1980), but the biochemical properties
`of this enzyme have not been well characterized. Brandon 50
`and Shugar (1980) suggest that salivary ribonuclease is
`pancreatic ribonuclease-like, but this has not been estab(cid:173)
`lished.
`At least in part as result of its instability RNA is often
`considered as an unsuitable analyte for diagnosis or detec(cid:173)
`tion. In the case of RNA viruses, methods have been devised
`for detection that do not require direct detection of RNA. For
`example, liquid culturing systems are used to 'grow up'
`sufficient quantities of virus/bacteria to confirm a diagnosis.
`Bacterial infection is typically diagnosed by direct staining
`and microscopic examination of samples. Electron micros(cid:173)
`copy is also used to identify bacteria and virus containing
`samples. In serology, diagnosis may be accomplished by
`detection of antibodies directed against pathogens ( e.g.
`viruses, bacteria, parasites) in blood serum by employing
`indirect fluorescent antibody testing and enzyme-linked
`immunosorbent assays
`
`5
`
`2
`Reverse transcriptase PCR (RT-PCR) procedures are sen(cid:173)
`sitive for detecting pathogens, and in some cases before the
`onset of symptoms. Rapid viral diagnosis will become
`increasingly critical, both for the control of epidemics and
`for the management of patients with viral infections. Cur(cid:173)
`rently, an immunofluorescence assay (IFA) is considered the
`"gold standard" for the detection of SARS-CoA infection.
`However, this test requires culturing of infectious SARS
`virus in laboratories with biosafety level 3 (BSL-3) facilities
`10 by well-trained technician personnel. Hence, there is a need
`for a more convenient, economical, and low-risk method for
`collecting and processing infectious clinical specimens.
`RNA can be extracted from most, if not all, cell types in
`the human body (except erythrocytes) and from a variety of
`15 cell-containing bodily fluids and/or secretions as well as
`tissues. In some cases, it is also be desirable to be able to
`obtain RNA from other sources, including feces, urine,
`cerebral spinal fluid, animal tissues, bone marrow aspirates,
`plants, plant extracts, microorganisms, virus, soil samples,
`20 sewage, wastewater, and/or foodstuffs (including milk).
`Typically, once a RNA-containing sample is collected, it
`must either be frozen ( e.g., with liquid nitrogen) or quickly
`transported in the unfrozen state at 4° C. to a laboratory for
`extraction of RNA. The requirement for rapid transportation
`25 and/or the requirement of freezing may be problematic in
`terms of cost and storage space. Additionally, in the case of
`remote locations and/or large-scale sample collection, rapid
`transportation and/or freezing may not be feasible. Impor(cid:173)
`tantly, rapid processing/testing of clinical samples may not
`30 be feasible during an epidemic; back-logged samples will
`likely degrade over time and/or under sub-optimal storage
`conditions. A simpler procedure for collecting RNA in a
`form that would not require the sample be frozen or trans(cid:173)
`ported immediately to a laboratory including equipment
`35 such as freezers, refrigerators, centrifuges, etc., would be
`desirable.
`As noted above, there are a variety of cellular sources of
`RNA. Cells from the oral cavity are conveniently obtained
`from samples of saliva. Saliva can be collected 'passively'
`by spitting and/or 'actively' with the aid of implements ( e.g.,
`swabs). Nasal mucosa! samples are conveniently obtained
`and are a rich source or epithelial and immune cells (e.g.,
`lymphocytes). This procedure is not as invasive compared
`to, for example, taking of venous blood and a simple
`45 procedure based on saliva would permit self-collection by
`individuals with essentially no prior training. However, once
`collected, the time that useable RNA can be recovered may
`be limited because of the presence of ribonucleases in most
`tissues and bodily fluids.
`With the increasing use of nucleic acid-based testing in
`human and veterinary medicine and in research, there is a
`need for compositions and methods that would allow RNA
`to be reliably recovered from bodily fluids and/or secretions
`and tissues. Desirably, it should be possible to be able to
`55 store the collected bodily fluid or bodily tissue at ambient
`temperature for prolonged periods of time, for example
`several days or weeks. For example, this would be advan(cid:173)
`tageous where the bodily sample or bodily tissue needs to be
`shipped to a distant location for purification and analysis,
`60 especially in the absence of refrigeration or freezing.
`Cationic compounds, such as tetradecyltrimethylammo(cid:173)
`nium oxalate, have been used previously as a component in
`acids.
`in purification of nucleic
`solutions
`used
`US20020146677
`includes
`tetradecyltrimethylanimonium
`65 oxalate plus tartaric acid to stabilize nucleic acid in blood.
`However, cationic compounds, including tetradecyltrimeth(cid:173)
`ylammonium oxalate, have been found to be unsatisfactory
`
`Page 21
`
`

`

`US 10,000,795 B2
`
`3
`in terms of ease of use and long term stability of RNA. It has
`been found that once the cationic detergent is bound to
`nucleic acids, the nucleic acids are difficult to dissolve.
`In addition, it would be desirable for the amount of RNA
`in the collected sample to be sufficiently large to allow for 5
`the detection of low copy number RNA species such as
`messenger RNA and some viruses.
`This background information is provided for the purpose
`of making known information believed by the applicant to
`be of possible relevance to the present invention. No admis- 10
`sion is necessarily intended, nor should be construed, that
`any of the preceding information constitutes prior art against
`the present invention.
`
`SUMMARY OF THE INVENTION
`
`4
`FIG. 6 is photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RNA in saliva stored in a composition of the
`present invention over a range of SDS concentrations;
`FIG. 7 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RNA in saliva when stored at room temperature
`compared to 37° C., using a composition of the present
`invention;
`FIG. 8 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RNA in saliva stored at room temperature in a
`composition of the present invention;
`FIG. 9 is a photograph of an ethidium bromide-stained,
`15 transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RNA in saliva stored at room temperature in a
`composition of the present invention;
`FIG. 10 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro-
`20 phoresis of RNA samples stored in compositions of the
`present invention and heated at various temperatures sub(cid:173)
`sequent to storage at room temperature;
`FIG. llA-B are photographs of ethidium bromide(cid:173)
`stained, transilluminated agarose gels showing the results of
`25 electrophoresis of RNA samples stored in compositions of
`the present invention and heated at various temperatures
`subsequent to storage at room temperature; subject 1 (FIG.
`llA) and subject 2 (FIG. 11B);
`FIG. 12 is a photograph of an ethidium bromide-stained,
`30 transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RNA samples stored at room temperature for
`1016 days at room temperature in a composition of the
`present invention;
`FIG. 13 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RT-PCR products;
`FIG. 14A-D are photographs of ethidium bromide-
`stained, transilluminated agarose gels showing the results of
`electrophoresis of RNA samples from saliva stored at the
`indicated temperature for 1 week (FIG. A-B) and 8 weeks
`(FIG. C-D); and
`FIG. 15 is a diagram depicting steps that may be followed
`to collect saliva from a subject.
`
`An object of the present invention is to provide a com(cid:173)
`position and method for prolonged storage of RNA from
`bodily fluids and/or tissues at room temperature, which
`compositions and methods further facilitate extraction of the
`RNA in as high a yield and as nearly intact state as is
`possible.
`In accordance with one aspect of the present invention
`there is provided a composition for extracting and storing
`ribonucleic acid from a sample such that the ribonucleic acid
`within said sample remains stable at room temperature, said
`composition comprises: an anionic detergent; and a buffer(cid:173)
`ing agent at a pH of about 5 to about 8.2; wherein said
`composition stabilizes said ribonucleic acid at room tem(cid:173)
`perature.
`In accordance with another aspect of the present invention
`there is provided a method for preserving ribonucleic acid
`from a biological sample comprising the steps of: a. obtain(cid:173)
`ing the sample from a subject; b. contacting said sample with 35
`a composition comprising an anionic denaturing agent and a
`buffering agent at a pH of about 5 to about 8.2 to form a
`mixture; c. storing the mixture at room temperature; and d.
`heating the mixture at greater than or about equal to 50° C.
`prior to subsequent processing, wherein said composition 40
`stabilizes said ribonucleic acid at room temperature.
`In accordance with another aspect of the present invention
`there is provided a RNA storage kit, comprising: a. a
`composition according to the present invention.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`45
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`FIG. 1 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of nucleic acids from saliva stored in compositions
`of the present invention and stored in Oragene™;
`FIG. 2 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of nucleic acids from saliva stored in Oragene™
`FIG. 3 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RNA samples stored in compositions having a
`range of pH's;
`FIG. 4A-C are photographs of ethidium bromide-stained,
`transilluminated agarose gels showing the results of elec(cid:173)
`trophoresis of RNA combined with a cell-free fraction of
`saliva; subject 1 (FIG. 4A), subject 2 (FIG. 4B), and subject
`3 (FIG. 4C);
`FIG. 5 is a photograph of an ethidium bromide-stained,
`transilluminated agarose gel showing the results of electro(cid:173)
`phoresis of RNA combined with a cell-free fraction of saliva
`over a range of pH's;
`
`As will be described in more detail below, the present
`invention relates to compositions and methods for prolonged
`50 storage, and extraction, of ribonucleic acid (RNA) from
`bodily fluids such as saliva, nasal secretions and/or tissues,
`wherein the RNA in the resulting composition remains
`stable at room temperature for extended periods of time.
`The term "about", as used herein, refers to +/-10% of the
`55 stated value or a chemical or obvious equivalent thereof.
`The term "bodily fluid", as used herein, refers to a
`naturally occurring fluid from a human or an animal, and
`includes, but is not limited to saliva, sputum, serum, plasma,
`blood, pharyngeal, nasal/nasal pharyngeal and sinus secre-
`60 tions, urine, mucous, gastric juices, pancreatic juices, bone
`marrow aspirates, cerebral spinal fluid, feces, semen, prod(cid:173)
`ucts oflactation or menstruation, cervical secretions, vaginal
`fluid, tears, or lymph.
`The terms "bodily tissue" or "tissue", as used herein, refer
`65 to an aggregate of cells usually of a particular kind together
`with their intercellular substance that form one of the
`structural materials of a plant or an animal and that in
`
`Page 22
`
`

`

`US 10,000,795 B2
`
`6
`
`5
`animals include connective tissue, epithelium, mucosa!
`membrane, muscle tissue, and nerve tissue, and the like.
`The term "Ct value", as used herein, is as defined in the
`Operator Manual for our Rotor-Gene™ 6000 (real-time
`genetic amplification detection system; manufactured by
`Corbett Life Science) and refers to the fractional cycle
`number at the point where the am