WO2010083844A1 - Methods and uses for rna extract and storage - Google Patents

Methods and uses for rna extract and storage Download PDF

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Publication number
WO2010083844A1
WO2010083844A1 PCT/DK2010/050018 DK2010050018W WO2010083844A1 WO 2010083844 A1 WO2010083844 A1 WO 2010083844A1 DK 2010050018 W DK2010050018 W DK 2010050018W WO 2010083844 A1 WO2010083844 A1 WO 2010083844A1
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rna
acid
sodium
composition
sample
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PCT/DK2010/050018
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French (fr)
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Limei Meng Okkels
Nikolaj Dam Mikkelsen
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Quantibact A/S
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • RNA performs a variety of roles in the cell. It acts as mRNA, tRNA and rRNA in the process of translating genetic information in DNA into proteins. Moreover, small RNAs of bacteria and microRNAs of higher organisms such as mammals act as post-transcriptional gene regulators. In clinical laboratories, RNA based analysis is becoming an important tool for disease diagnostics. Thus, methods enabling quantitative and qualitative studies of the RNA molecules in a cell are of immense importance and of relevance for basic research, development of therapeutics as well as for monitoring and diagnostics.
  • RNA-based analytic methods are the purity and integrity of the RNA sample employed.
  • the integrity of a RNA sample can easily be compromised by the biological and chemical instability of RNA.
  • RNA when an RNA-containing sample is collected, RNA is typically preserved by immediate freezing and storage at -2O 0 C or -8O 0 C until RNA extraction takes place in the laboratory.
  • This is often a challenge for handling clinical samples, such as urine, blood, away from an analysis laboratory, as these samples can first be processed when they arrive the laboratory, and the transportation, which usually take place at room temperature, can take days.
  • it is critical to preserve the integrity/stability of the RNA Just by simple handling, the RNA sample may be contaminated by RNases of the investigator, wherefore utmost care is needed for RNA work
  • US, 5, 973, 137 describes a kit for isolating RNA comprising instruction means for isolating substantially undegraded RNA from a biological sample and a cell lysis reagent including 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.
  • the anionic detergent is preferably present in an amount of about 0,5-3,0 %. Such concentration of anionic detergents is generally detrimental to enzymatic activity, because enzymes are denatured by the detergent.
  • US, 5, 973, 137 describes a process for RNA isolation from yeast cells, wherein a lytic enzyme reagent is used.
  • the lytic enzyme reagent comprises a lytic enzyme that digests beta-l,3-glucose polymers that are contained in yeast cell walls. It is also mentioned that the lytic reagent may be used for RNA isolation from gram- positive bacteria, but only RNA extraction from yeast is exemplified.
  • cells are resuspended in a cell suspension reagent, where after a lytic enzyme reagent is added.
  • the pH of the cell suspension reagent preferably has a pH of about 7-8,5, and more preferably between 7,5-8.
  • the pH of the lytic enzyme reagent is between 7,5 and 8,2.
  • enzymatic lysis is done at a pH close to neutral, and without the presence of anionic detergents. This is in accordance with the general expectation that lytic enzymes would be inactivated by anionic detergents and/or acidic pH; however, under the above conditions RNA may be degraded during the time when cells are treated with lytic enzymes.
  • WO2008/040126 describes a composition for extracting and storing RNA from a sample such that the RNA within the sample remains stable at room temperature, said composition comprising: an anionic detergent; and a buffering agent at a pH of about 5,2 to about 8, wherein said composition stabilizes said RNA at room temperature.
  • the pH is in the range of 5,0-8,2; 5,1 - 7,0; 5,5 - 7,5; 6,5 - 7,0 or 6,8.
  • the anionic detergent is preferably SDS in a concentration range of about 0,5% to about 8%. It is described that once the SDS concentration is diluted below a certain concentration, e.g. below 0,5% SDS, the RNA within the sample may be substantially degraded. Thus, to preserve stability of the RNA, the SDS concentration should be above 0,5% SDS. In the examples, an SDS concentration between 4% and 16% is used and the pH is typically 6,6 or 6,8.
  • WO2008/040126 disclosed a composition that can stabilize and release the RNA from human cells in saliva, it did not disclose a composition that release RNA from cell-wall containing cells, e.g. gram positive bacteria, mycobacteria, yeast, fungi, and plant cells. It is known to skilled people in the art that cell-wall containing cells cannot be lysed by anionic detergent alone, additional means needs to be employed.
  • RNA protection reagents in particular RNA protection reagents that have low toxicity and are compatible with enzymatic processes, such as e.g. enzymatic lysis processes.
  • the present invention provides use of a composition for protecting RNA against degradation, said composition comprising an anionic detergent at concentration between 0,01% and 0,5% and a pH below 7. Said use is applicable for e.g. RNA storage and cell lysis.
  • Lanes 1-4 pH 7 (Sodium phosphate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively.
  • Lanes 5-8 pH 6.2 (Sodium phosphate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively.
  • Lanes 9-12 pH 6.4 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively.
  • Lanes 13-16 pH 5.7 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively.
  • Lanes 17 -20 pH 5.2 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively.
  • Lanes 21-24 pH 4.6 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively.
  • Lanes 1-3 pH 5.7 (Sodium acetate buffer); 0%, 0.24%, and 0.12% SDS, respectively.
  • Lanes 4-7 pH 5.2 (Sodium acetate buffer); 0%, 0.24%, 0.12%, and 0.06% SDS, respectively.
  • Lanes 8-12 pH 4.6 (Sodium acetate buffer); 0%, 0.24%, 0.12%, 0.06%, and 0.03% SDS, respectively.
  • Fig 3. rRNA preservation in Staphylococcus aureus cells in buffered urine, after have stored 4 days at room temperature. Lane 1 : 71% urine; lane 2: 38% urine; lane 3: 19% urine; Lane 4: 9.5% urine; lane 5 : 4.8% urine; lane 6: 0% urine.... etc.
  • Fig. 4. Detection of bacterial rRNA by capture assay.
  • Lane 1 0% SDS. Lane 2: 0.25% SDS. Lane3: 0.125% SDS. Lane 4: 0.06% SDS; Lane 5: 0.03% SDS. Lane 6: Total RNA from E. coli (Ambion)
  • composition for RNA protection Composition for RNA protection
  • a first aspect of the invention is the use of a composition for protecting RNA against degradation wherein said composition comprises
  • An anionic detergent at a concentration of between 0,01% and 0,5%, when the composition is mixed with a sample b.
  • a buffering agent keeping the pH below 7, when the composition is mixed with the sample.
  • reference will in general be to the pH and concentrations when the composition is mixed with a sample. In certain embodiments, reference may be to the composition before it is added to sample and this will be specifically mentioned for these embodiments.
  • the pH of the composition is below 6.
  • the pH is between 4 and 7, more preferably between 4,5 and 6 and most preferably between 5 and 6.
  • the concentration of the anionic detergent is between 0.01% to 0.5%, preferably 0.01 to 0.25%, more preferably, 0.01 to
  • the anionic detergent is preferably selected from the group consisting of alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, alpha sulfonyl fatty acids, alkyl phosphates, dioctyl sulfosuccinate, isethionates, alkyl ether sulfates, methyl sarcosines and the like.
  • suitable anionic detergents include amine dodecylbenzene sulfonate; ammonium capryleth sulfate; ammonium cumenesulfonate; ammonium dihydroxy stearate; ammonium dodecylbenzene sulfonate; ammonium laureth sulfate; ammonium laureth-12 sulfate; ammonium laureth- 30 sulfate; ammonium lauroyl sarcosinate; ammonium lauryl sulfate; ammonium lauryl sulfosuccinate; ammonium lignosulfonate; ammonium myreth sulfate; ammonium naphthalene sulfonate; ammonium nonoxynol-20 sulfate; ammonium nonoxynol-30 sulfate; ammonium nonoxynol-4 sulfate; ammonium nonoxynol-6 sul
  • the anionic detergent is selected from the group consisting of glycolic acid ethoxylate octyl ether; glycolic acid ethoxylate oleyl ether; glycolic acid ethoxylate lauryl ether; poly(ethylene glycol) 4-nonylphenyl 3- sulfopropyl ether potassium salt; glycolic acid ethoxylate 4-tert-butylphenyl ether; glycolic acid ethoxylate oleyl ether; glycolic acid ethoxylate oleyl ether; poly(ethylene glycol) n-alkyl 3-sulfopropyl ether potassium salt; glycolic acid ethoxylate 4-nonylphenyl ether; poly(ethylene glycol) n-alkyl 3-sulfopropyl ether potassium salt; sodium cholate hydrate; sodium deoxycholate; sodium taurodeoxycholate hydrate; sodium taurocholate; sodium cholate hydrate; sodium deoxycholate; sodium tau
  • the anionic detergent is selected from the group consisting of poly(ethylene glycol)4-nonphenyl 3-sulfopropyl ether potassium salt; poly(ethylene glycol) monolaurate, carrageenan lambda; polyoxyethylene(150)dinonylphenyl ether polyoxyethylene (Igepal ⁇ (R)> DM- 970); and nonyl nonoxynol-15 phosphate (Rhodafac RM710).
  • the anionic detergent is a salt of dodecyl sulfate, such as sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate. Most preferably, the anionic detergent is SDS.
  • the anionic detergent is not a salt of dodecyl sulfate, such as sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate.
  • dodecyl sulfate such as sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate.
  • the buffering agent may be any agent capable of buffering the pH to the desired value.
  • the buffering agent is selected from the group consisting of sodium cyclohexane diaminetetraacetate (CDTA), N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid (BES), A- (2-Hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES), acetic acid or acetate (e.g.
  • the buffering agent has a pKa at 25 0 C of between 4.0 and 8.0, more preferably between 4.5 and 7.5, most preferably between 4.75 and 7.
  • the composition may further include a chelating agent capable of chelating a divalent cation. Chelating agents may be included because divalent cations are required for optimal activity of many RNases. I.e. when chelating agents are included, many RNases will be less active. Thus, in one embodiment, the composition comprises chelating agents such as EDTA, Citrate or CDTA.
  • the composition without any chelating agents is capable of protecting RNA against degradation. Therefore, in another embodiment, the composition does not comprise a chelating agent such as EDTA, citrate or CDTA.
  • a chelating agent such as EDTA, citrate or CDTA.
  • examples of enzymes could be proteinase, lysozyme, Mutanolysin and Lysostaphin.
  • the composition comprises urea.
  • the concentration of urea is preferably below IM.
  • the concentration is below 10OmM; 1OmM; 5 mM, 4 mM, 3 mM, 2 mM, ImM; 0,1 mM; 0,0ImM and 0,001 mM respectively .
  • the composition may also comprise a chaotropic agent for denaturation of proteins and other cellular components.
  • exemplary chaotropic agents are guanidine thiocyanate, sodium thiocyanate, guanidinium chloride, sodium iodide, potassium iodide and urea.
  • Preferred concentration of chaotropic agents are more than 1 M, more than 2 M, more than 3 M and between IM and 5 M.
  • the composition does not comprise chaotropic agents at a concentration above 100 mM; above 10 mM; 1 mM; 0,1 mM; 0,01 mM and 0,001 mM. In one embodiment, the composition does not comprise a chaotropic agent.
  • the composition is a RNA storage solution used for improving storage stability of RNA.
  • the RNA may be purified, i.e. substantially free of other macromolecules of the cell or the RNA may still be present in a cell or in a virus (as will be further outlined below).
  • the RNA storage solution is a stock solution for dilution for between dilution between 1 and 20 times.
  • RNA storage solution 1 volume of a 10 X concentrated stock solution of the RNA storage solution may be added to 9 volumes of RNA solution. If the RNA has been precipitated and dried, a IX concentrated RNA storage solution may be used for resuspension and subsequent storage.
  • the RNA storage solution may be added to the bacteria containing sample (e.g. urine) or bacterial culture at appropriate amounts, where after the sample can be stored with reduced or no degradation of the RNA.
  • sample e.g. urine
  • sample may be stored at room temperature (between 18 and 28 0 C), between 0 and 5 0 C or below 0 0 C.
  • the sample may be heated to more than 80 0 C or more preferably more than 94 ° C degrees before storage.
  • the RNA storage solution makes handling of samples comprising bacteria for RNA purification much easier since using the RNA storage solution lessens the requirements for fast transportation to the laboratory for either purification or storage in the freezer and later purification.
  • RNA storage solution may be provided as any appropriate stock solution. However, stock solutions that are intended for the following dilutions are preferred: 1, 2, 4, 5 and 10.
  • the composition is a suspension solution for suspending (dissolving) precipitated RNA or for suspending RNA containing cells or virus present in the sample.
  • the cells are typically single cell organisms such as yeast and bacteria. However, it may also be used for plants cells or tissue cells, e.g. when the tissue or plant has been grinded.
  • the composition is a cell lysis solution for lysing RNA containing cells or virus present in the sample.
  • the cell lysis solution may also comprise a lytic enzyme to facilitate lysis.
  • the lytic enzyme may be added to the cell lysis solution prior to use of the cell lysis solution. I.e. the lytic enzyme could be supplied in a separate vial for addition to the cell lysis solution.
  • the lytic enzyme is a peptidoglucan degrading enzyme, e.g. lysozyme, mutanolysin (a N-Acetyl Muramidase that cleaves the N-acetylmuramyl- ⁇ (l-4)-N- acetylglucosamine linkage of the bacterial cell wall polymer peptidoglycan- polysaccharide), lysostaphin (a zinc endopeptidase that cleaves the polyglycine cross-links in the peptidoglycan layer of the cell wall), labiase (contains ⁇ -N- acetyl-D-glucosaminidase and lysozyme activity), achromopeptidase (a lysyl endopeptidase), or a combination peptidoglucan degrading enzyme.
  • lysozyme a N-Acetyl Muramidase that cleaves the N-acetylmuramyl
  • the lytic enzyme may also be a protein degrading enzyme, e.g. proteinase K.
  • the lytic enzyme may also be fungi cell wall glucan degrading enzyme.
  • the lytic enzyme is a peptidoglucan degrading enzyme
  • the cell lysis solution is particular suited for lysis of bacteria.
  • the cells as mentioned in any of the previous embodiments may be any cells. In a preferred embodiment they are selected from the group consisting of bacteria, yeast, fungi, plant cells and mammalian cells.
  • the bacteria may be gram-negative such as e.g. E.coli, Salmonella typhimunium, Klebsiella pneumoniae, a gram positive such as e.g. Staphlococcus aureus, Staphyloccocus spp, Stapylococcus saprophytic, Enterococcus faecalis, , or a mycobacterium.
  • the sample is urine collected from a mammal, preferably from a human.
  • the cells may be cells of the mammal to be used e.g. for diagnosis or detection.
  • the cells may also be bacteria or fungi.
  • the sample may also be or comprise cells from a tissue sample or other body fluids such as whole blood, cerebrospinal fluid, plasma, saliva, semen, serum or synovial fluid.
  • the sample is heated to at least 50 ° C to facilitate cell lysis, preferably heating is done after the sample has been mixed with the composition.
  • the cell lysis solution When used for lysis of bacteria, it preferably comprises anionic detergent at a concentration between 0,01% and 0,5% and a buffering agent keeping the pH between 4 - 7.
  • the composition is a RNA or DNA precipitation solution.
  • the composition enables DNA or RNA precipitation while protecting the RNA from degradation.
  • the composition comprises salt at concentration of at least 300 mM.
  • 2 volumes ethanol or 1 volume isopropanol is added to facilitate precipitation.
  • the salt is preferably sodium or potassium for DNA precipitation.
  • Such composition can e.g. be used to precipitate DNA during phenol extraction. It can also be used to precipitate DNA using centrifugation. As will clear, precipitation of DNA is one step towards RNA purification.
  • the salt is preferably lithium and the composition may be used during extraction or centrifugation as mentioned above for DNA precipitation.
  • a second aspect of the invention is a method comprising contacting the sample with a composition as described in the first aspect of the invention. Preferred embodiments of the method are described in the first aspect. They are briefly mentioned in the following.
  • the method is a method of storing RNA in a sample.
  • the method is a method of precipitating RNA or DNA.
  • the method is a method of suspending cells comprising RNA or suspending precipitated RNA.
  • the method is a method of lysing cells or virus containing RNA.
  • the composition is preferably a cell lysis solution as outlined in the first aspect.
  • the methods may further comprise a step of selected from the group consisting of: a. Phenol extraction and RNA precipitation b. Immobilization c. Differential centrifugation (cesium chloride) d. Capturing using a capture probe
  • RNA may be further processed e.g. by any of steps a-d just mentioned before being employed in detection methods. Alternatively it may be used directly in detection methods, such as cDNA preparation, PCR or qPCR microarray analysis, northern blotting, dot blotting, luminex etc.
  • a more specific embodiment of the cell lysis method comprises a) Storing the sample in RNA storage solution described in the earlier section at room temperature; b) treating the sample with lytic enzymes (optional); c) add anionic detergent to at least 0.5%, d) or add other lytic agents; and optionally e) heating the sample to at least 5O 0 C for 5 - 10 min to facilitate cell lysis
  • the cells are gram positive bacteria, and step b is non-optional.
  • a third aspect of the invention is a kit comprising a solution selected from the group consisting of:
  • a cell suspension solution as described in the first aspect b.
  • c. A RNA precipitation solution as described in the first aspect.
  • d. A RNA storage buffer according as described in the first aspect.
  • the kit of comprises a. A cell suspension solution as described in the first aspect. b. A cell lysis solution according as described in the first aspect. c. Instructions for use
  • the kit comprises a. A cell suspension solution as described in the first aspect. b. A cell lysis solution according as described in the first aspect. c. A RNA storage buffer according as described in the first aspect. d. Instructions for use In another embodiment the kit comprises a. A cell suspension solution as described in the first aspect. b. A cell lysis solution according as described in the first aspect. c. A RNA precipitation solution as described in the first aspect. d. A RNA storage buffer as described in the first aspect. e. Instructions for use
  • Urine sam ple Urine specimens from hospital patients were tested for bacterial growth at Clinical Microbiological laboratory, Hvidovre Hospital, Copenhagen. Fifty-three of the specimens lacking visible bacterial growth on nonselective growth medium were pooled, and the pH was determined to be 6.5, measured by a pH meter. The urine sample was stored in 3.5 ml aliquots at -2O 0 C,
  • Urine RNase activity was studied by the following procedure.
  • the urine sample was buffered with either Sodium Phosphate or Sodium Acetate buffer to pH range 5.2 - 7.0, and SDS was added to a range of 0 - 0.1%.
  • the final samples were of 12 ⁇ l volume and containing 58% of urine, 42 mM of Sodium Phosphate buffer or 42 mM Sodium Acetate buffer, 2 ⁇ g of purified E. coli total RNA (Ambion).
  • the samples were incubated at room temperature for 15 min. Reactions were stopped by adding 3 ⁇ l of 10% SDS to each sample, and RNA degradation was analysed by electrophoresis on a 1.3% agarose gel in TAE runing buffer.
  • E. coli culture E. coli strain ATCC 25922 was cultured by the following procedure. Seven ml serum broth was inoculated with frozen £ coli stock and incubated at 37 0 C overnight without agitation. One ml of the overnight culture was transferred to 9 ml fresh serum broth and subsequently incubated at 37 0 C for 3 1 /2 hr without agitation. £ coli cells were harvested in 1 ml aliquots by centrifuging at 9000 rpm for 2 min; the supernatant was discarded and the cell pellets were stored at -2O 0 C.
  • the urine sample (described in Example 1) was buffered with either Sodium Phosphate or Sodium Acetate buffer to pH 4.6 to 7.0; SDS was added to a range of 0 to 0.24%. £ coli was spiked into the buffered urine to approximately 5x 10 8 CFU/ml. The final samples were of 105 ⁇ l volume and containing 67% of urine, 48 mM of Sodium Phosphate buffer or 48 mM Sodium Acetate buffer. The samples were stored at room temperature.
  • RNA preservation 10 ⁇ l of each sample was lysed by adding 2 ⁇ l of 10% SDS, and the preservation of 16S and 23S rRNA was analysed by electrophoresis on a 1.3% agarose gel in TAE runing buffer
  • Gram positive bacterial culture Staphylococcus aureus (ATCC 29212), was cultured according to the procedure similar to that of E. coli culture described in Example 1. The bacterial cells were harvested in 1 ml aliquots and the cell pellets were stored at -2O 0 C as described in example 1. Human urine sample was buffered with sodium acetate (final concentration 48 mM) to pH 5.7, and spiked with Staphylococcus aureus. SDS was added to 0.12%. The final sample contains 71%, 38%, 19%, 9.5% , 4.8% or 0% urine (Fig. 3).
  • the samples were stored at room temperature for 4 days.
  • the samples were treated with a mixture of Hen Egg lysozyme (Fluka), Lysostaphin (Sigma), and mutanolysin (Sigma), in a final volume of 29 ul containing 0-62% urine, 0.1% SDS, 6 U mutanolysin, 6 U lysostaphin, 0.15 mg lysozyme, 43 mM Sodium Acetate buffer, with a final pH of 5.7.
  • the samples were incubated at room temp for 30 min, and then SDS was added to 2%. Five ⁇ l of each lysates were analysed, by electrophoresis on a 1.3% agarose gel in TAE runing buffer.
  • the urine sample (described in Example 1) was buffered with Sodium Acetate buffer to pH 5.0; SDS was added to 2%. £ coli was spiked into the buffered urine to approximately 5x 10 8 CFU/ml. The final samples were of 140 ⁇ l volume and containing 7% of urine, 45 mM Sodium Acetate buffer. The above mixture was incubated at room temperature for 15 min, and then incubated at 95 0 C for 5 min. This total lysate was diluted in a 4-fold serial dilution in H 2 O, and used directly in the capture assays below.
  • CATCGTTTACGGCGTGGACTACCAGGG was coupled to MagPlexTM-C Magnetic Carboxylated Microspheres (MC10015-04, Luminex Corporation, USA) by the carbodiimide coupling method provided by the manufacture.
  • the capture assays were performed in a 96-well microwell plate, and each assay contains 5ul (contains approximate 2500 beads) of the beads carrying capture probe, 10 ul of rRNA sample (see the scheme in Table 1), 60 ul hybridization buffer (10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, IM NaCI), 25 ul of biotin labeled detection probe (lOuM ) (EC0766: CATCGTTTACGGCGTGGACTACCAGGG).
  • the plate was incubated at 65 0 C for 25 min, and then the beads were washed 3 times with 100 ul Washing buffer (10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, 250 mM NaCI).
  • 100 ul of the Reporter Mix was added to each sample well (the detection mix contains 2.5 ⁇ g/ml of Strptavidin-R-PhycoErythrin (Invitrogen), 100 ⁇ g/ml BSA, 10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, and 250 mM NaCI), and subsequently incubated for 15 min at 25 0 C.
  • the beads were washed 3 times with 100 ul Washing buffer ((10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, 250 mM NaCI), and then resuspended in 100 ⁇ l of 0.5xTMAC buffer. Eighty ⁇ l_ of the beads were analysed on the Luminex analyzer, StarStation 3.0, according to the system manual (results are shown in Table IB). Ribosomal RNA from E. coli MRE600 (Roche Applied Science, cat. Nr. 10206938001, 4 ⁇ g/ ⁇ l) was used as control rRNA.
  • Human urine sample was buffered with Sodium Acetate buffer to pH 5.2.
  • the final samples were of 100 ⁇ l volume and containing 70% of urine, 50 mM Sodium Acetate buffer and 0,0.03%, 0.06%, 0.125%, or 0.25% SDS.
  • One tube of the E.coli pellet from example 2 was resuspended in 65 ⁇ l of sterile H 2 O, and 5 ⁇ l of the cell suspension was added to each of the samples above. The samples were stored at room temperature for 8 days.
  • nucleic acids were purified by the geneMAg-RNA/DNA kit according to manufacturer's instruction, except that smaller volumes of the magnetic beads and lysis/binding buffer were used (25 ⁇ l of magnetic beads, and 500 ⁇ l of the buffer). The nucleic acids were eluted in 50 ⁇ l of DEPC H2O; 15 ⁇ l of each preparation were analysed by electrophoresis on a 1.3% agarose gel in TAE runing buffer (results are shown in Fig.4)

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Abstract

The present invention relates to compositions and uses that protect RNA against degradation. Thus, the invention provides use of a composition for protecting RNA against degradation, said composition comprising an anionic detergent at concentration between 0,01% and 0,5% and a pH below 7. Said use is applicable for e.g. RNA storage and cell lysis.

Description

Methods and uses for RNA extraction and storage
Background
RNA performs a variety of roles in the cell. It acts as mRNA, tRNA and rRNA in the process of translating genetic information in DNA into proteins. Moreover, small RNAs of bacteria and microRNAs of higher organisms such as mammals act as post-transcriptional gene regulators. In clinical laboratories, RNA based analysis is becoming an important tool for disease diagnostics. Thus, methods enabling quantitative and qualitative studies of the RNA molecules in a cell are of immense importance and of relevance for basic research, development of therapeutics as well as for monitoring and diagnostics.
Critical factors for RNA-based analytic methods are the purity and integrity of the RNA sample employed. The integrity of a RNA sample can easily be compromised by the biological and chemical instability of RNA.
Thus, when an RNA-containing sample is collected, RNA is typically preserved by immediate freezing and storage at -2O0C or -8O0C until RNA extraction takes place in the laboratory. This is often a challenge for handling clinical samples, such as urine, blood, away from an analysis laboratory, as these samples can first be processed when they arrive the laboratory, and the transportation, which usually take place at room temperature, can take days. The longer the sample is stored before extraction, the more degraded the RNA may be. Also after RNA extraction, it is critical to preserve the integrity/stability of the RNA. Just by simple handling, the RNA sample may be contaminated by RNases of the investigator, wherefore utmost care is needed for RNA work
Therefore, there is a need for improved compositions and methods for RNA protection during especially sample transportation, storage, and cell lysis at room temperature.
US, 5, 973, 137 describes a kit for isolating RNA comprising instruction means for isolating substantially undegraded RNA from a biological sample and a cell lysis reagent including 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. The anionic detergent is preferably present in an amount of about 0,5-3,0 %. Such concentration of anionic detergents is generally detrimental to enzymatic activity, because enzymes are denatured by the detergent.
US, 5, 973, 137 describes a process for RNA isolation from yeast cells, wherein a lytic enzyme reagent is used. The lytic enzyme reagent comprises a lytic enzyme that digests beta-l,3-glucose polymers that are contained in yeast cell walls. It is also mentioned that the lytic reagent may be used for RNA isolation from gram- positive bacteria, but only RNA extraction from yeast is exemplified. In the method as described in the specification, cells are resuspended in a cell suspension reagent, where after a lytic enzyme reagent is added. Importantly, the pH of the cell suspension reagent preferably has a pH of about 7-8,5, and more preferably between 7,5-8. The pH of the lytic enzyme reagent is between 7,5 and 8,2. Thus, enzymatic lysis is done at a pH close to neutral, and without the presence of anionic detergents. This is in accordance with the general expectation that lytic enzymes would be inactivated by anionic detergents and/or acidic pH; however, under the above conditions RNA may be degraded during the time when cells are treated with lytic enzymes.
WO2008/040126 describes a composition for extracting and storing RNA from a sample such that the RNA within the sample remains stable at room temperature, said composition comprising: an anionic detergent; and a buffering agent at a pH of about 5,2 to about 8, wherein said composition stabilizes said RNA at room temperature.
The pH is in the range of 5,0-8,2; 5,1 - 7,0; 5,5 - 7,5; 6,5 - 7,0 or 6,8.
The anionic detergent is preferably SDS in a concentration range of about 0,5% to about 8%. It is described that once the SDS concentration is diluted below a certain concentration, e.g. below 0,5% SDS, the RNA within the sample may be substantially degraded. Thus, to preserve stability of the RNA, the SDS concentration should be above 0,5% SDS. In the examples, an SDS concentration between 4% and 16% is used and the pH is typically 6,6 or 6,8. Although WO2008/040126 disclosed a composition that can stabilize and release the RNA from human cells in saliva, it did not disclose a composition that release RNA from cell-wall containing cells, e.g. gram positive bacteria, mycobacteria, yeast, fungi, and plant cells. It is known to skilled people in the art that cell-wall containing cells cannot be lysed by anionic detergent alone, additional means needs to be employed.
The acidic pH and high concentrations of SDS disclosed in WO2008/040126, however, would prohibit the application of the commercially available lytic enzymes for disrupting cell walls, as the said conditions deactivate/inhibit these enzyme activities.
Even in view of the above described references, there remains a need for RNA protection reagents, in particular RNA protection reagents that have low toxicity and are compatible with enzymatic processes, such as e.g. enzymatic lysis processes.
Sum mary of the invention The present invention provides use of a composition for protecting RNA against degradation, said composition comprising an anionic detergent at concentration between 0,01% and 0,5% and a pH below 7. Said use is applicable for e.g. RNA storage and cell lysis.
Disclosure of the invention
Brief description of the drawings
Fig 1. Inhibition of Human Urine RNase by low concentrations of SDS. Lanes 1-4: pH 7 (Sodium phosphate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively. Lanes 5-8: pH 6.2 (Sodium phosphate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively. Lanes 9-12: pH 6.4 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively. Lanes 13-16: pH 5.7 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively. Lanes 17 -20: pH 5.2 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively. Lanes 21-24: pH 4.6 (Sodium acetate buffer); 0%, 0.1, 0.05%, and 0.025% SDS, respectively.
Fig 2. rRNA preservation in E. coli cells in buffered urine, after have stored 10 days at room temperature. Lanes 1-3: pH 5.7 (Sodium acetate buffer); 0%, 0.24%, and 0.12% SDS, respectively. Lanes 4-7 : pH 5.2 (Sodium acetate buffer); 0%, 0.24%, 0.12%, and 0.06% SDS, respectively. Lanes 8-12: pH 4.6 (Sodium acetate buffer); 0%, 0.24%, 0.12%, 0.06%, and 0.03% SDS, respectively.
Fig 3. rRNA preservation in Staphylococcus aureus cells in buffered urine, after have stored 4 days at room temperature. Lane 1 : 71% urine; lane 2: 38% urine; lane 3: 19% urine; Lane 4: 9.5% urine; lane 5 : 4.8% urine; lane 6: 0% urine.... etc.
Fig. 4. Detection of bacterial rRNA by capture assay.
Lane 1 : 0% SDS. Lane 2: 0.25% SDS. Lane3: 0.125% SDS. Lane 4: 0.06% SDS; Lane 5: 0.03% SDS. Lane 6: Total RNA from E. coli (Ambion)
Composition for RNA protection
A first aspect of the invention is the use of a composition for protecting RNA against degradation wherein said composition comprises
a. An anionic detergent at a concentration of between 0,01% and 0,5%, when the composition is mixed with a sample b. A buffering agent keeping the pH below 7, when the composition is mixed with the sample.
It should be understand that the use of the first aspect may just as well be described as a method. When referring the pH and concentrations of various components of the composition, reference will in general be to the pH and concentrations when the composition is mixed with a sample. In certain embodiments, reference may be to the composition before it is added to sample and this will be specifically mentioned for these embodiments.
More preferably, the pH of the composition is below 6.
In an alternative embodiment, the pH is between 4 and 7, more preferably between 4,5 and 6 and most preferably between 5 and 6.
The concentration of the anionic detergent is between 0.01% to 0.5%, preferably 0.01 to 0.25%, more preferably, 0.01 to
0.15%, most preferably 0.02 to 0.1%.
The anionic detergent is preferably selected from the group consisting of alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, alpha sulfonyl fatty acids, alkyl phosphates, dioctyl sulfosuccinate, isethionates, alkyl ether sulfates, methyl sarcosines and the like.
Representative examples of suitable anionic detergents include amine dodecylbenzene sulfonate; ammonium capryleth sulfate; ammonium cumenesulfonate; ammonium dihydroxy stearate; ammonium dodecylbenzene sulfonate; ammonium laureth sulfate; ammonium laureth-12 sulfate; ammonium laureth- 30 sulfate; ammonium lauroyl sarcosinate; ammonium lauryl sulfate; ammonium lauryl sulfosuccinate; ammonium lignosulfonate; ammonium myreth sulfate; ammonium naphthalene sulfonate; ammonium nonoxynol-20 sulfate; ammonium nonoxynol-30 sulfate; ammonium nonoxynol-4 sulfate; ammonium nonoxynol-6 sulfate; ammonium nonoxynol-9 sulfate; ammonium oleic sulfate; ammonium perfluorooctanoate; ammonium stearate; ammonium xylenesulfonate; butyl naphthalene sulfonate; butyl phosphate; calcium dodecylbenzene sulfonate; calcium stearoyl lactylate; calcium tetrapropylenebenzene sulfonate; capryleth-9 carboxylic acid; cetyl phosphate; cumene sulfonic acid; DEA-cetyl phosphate; DEA-dodecylbenzene sulfonate; DEA-lauryl sulfate; deceth-4 phosphate; diammonium lauryl sulfosuccinate; diammonium stearyl sulfosuccinamate; diamyl sodium sulfosuccinate; dicyclohexyl sodium sulfosuccinate; dihexyl sodium sulfosuccinate; diisobutyl sodium sulfosuccinate; dilaureth-7 citrate; dimethiconol; dinonoxynol-4 phosphate; dioctyl ammonium sulfosuccinate; dioctyl sodium sulfosuccinate; disodium cetearyl sulfosuccinamate; disodium cocamido MEA- sulfosuccinate; disodium cocamido PEG-3 sulfosuccinate; disodium deceth-6 sulfosuccinate; disodium decyl diphenyl ether disulfonate; disodium dodecyloxy propyl sulfosuccinamate; disodium isodecyl sulfosuccinate; disodium laneth-5 sulfosuccinate; disodium lauramido DEA-sulfosuccinate; disodium lauramido MEA- sulfosuccinate; disodium laureth sulfosuccinate; disodium lauryl sulfosuccinate; disodium myristamido MEA-sulfosuccinate; disodium oleamido MEA- sulfosuccinate; disodium oleamido PEG-2 sulfosuccinate; disodium oleth-3 sulfosuccinate; disodium PEG-4 cocamido MIPA sulfosuccinate; disodium ricinoleamido MEA-sulfosuccinate; disodium stearyl sulfosuccinamate; disodium undecylenamido MEA-sulfosuccinate; ditridecyl sodium sulfosuccinate; dodecenylsuccinic anhydride; dodecyl diphenyl ether disulfonic acid; dodecyl diphenyloxide disulfonic acid; dodecylbenzenesulfonic acid; glyceryl dioleate SE; glyceryl distearate SE; glyceryl ricinoleate SE; glyceryl stearate citrate; glyceryl stearate SE; glycol stearate SE; hexyl phosphate; isopropyl phosphate; isopropylamine dodecylbenzenesulfonate; isosteareth-2 phosphate; isotrideceth-3 phosphate; isothdeceth-6 phosphate; laureth-1 phosphate; laureth-12 carboxylic acid; laureth-3 phosphate; laureth-4 phosphate; laureth-6 phosphate; laureth-7 citrate; laureth-9 phosphate; lauryl phosphate; lithium lauryl sulfate; magnesium laureth sulfate; magnesium PEG-3 cocamide sulfate; MEA-laureth phosphate; MEA-lauryl sulfate; MIPA-laureth sulfate; MIPA-lauryl sulfate; myristoyl sarcosine; naphthalene- formaldehyde sulfonate; nonoxynol-10 phosphate; nonoxynol-12 phosphate; nonoxynol- 3 phosphate; nonoxynol-4 phosphate; nonoxynol-4 sulfate; nonoxynol-6 phosphate; nonoxynol-7 phosphate; nonoxynol-8 phosphate; nonoxynol-9 phosphate; nonyl nonoxynol-10 phosphate; nonyl nonoxynol-15 phosphate; nonyl nonoxynol-7 phosphate; oleth-10 carboxylic acid; oleth-10 phosphate; oleth-3 carboxylic acid; oleth-4 phosphate; oleth-5 phosphate; oleth-6 carboxylic acid; oleth-7 phosphate; PEG-2 dilaurate SE; PEG-2 dioleate SE; PEG-2 distearate SE; PEG-2 laurate SE; PEG-2 oleate SE; PEG-2 stearate SE; PEG-9 stearamide carboxylic acid; potassium cetyl phosphate; potassium deceth-4 phosphate; potassium dodecylbenzene sulfonate; potassium isosteareth-2 phosphate; potassium lauroyl sarcosinate; potassium lauryl sulfate; potassium oleate; potassium oleic sulfate; potassium perfluorooctoate; potassium ricinoleic sulfate; PPG-2 laurate SE; PPG-2 oleate SE; PPG-2 stearate SE; PPG-5- ceteth-10 phosphate; propylene glycol laurate SE; propylene glycol oleate SE; propylene glycol ricinoleate SE; propylene glycol stearate SE; PVM/MA copolymer; sodium 2-ethylhexyl phosphate; sodium 2-ethylhexyl sulfate; sodium a olefin sulfonate; sodium allyloxy hydroxypropyl sulfonate; sodium behenoyl lactylate; sodium butoxyethoxy acetate; sodium butyl naphthalene sulfonate; sodium butyl oleate sulfate; sodium butyl oleate sulfonate; sodium butyl phosphate; sodium caproyl lactylate; sodium caprylyl sulfonate; sodium cetyl sulfate; sodium cumenesulfonate; sodium deceth sulfate; sodium decyl diphenyl ether sulfonate; sodium decyl sulfate; sodium dibutyl naphthalene sulfonate; sodium didodecylbenzene sulfonate; sodium diisooctyl sulfosuccinate; sodium diisopropyl naphthalene sulfonate; sodium dilaureth-7 citrate; sodium dinonyl sulfosuccinate; sodium dodecyl diphenyl ether disulfonate; sodium dodecyl diphenyloxide disulfonate; sodium dodecylbenzenesulfonate; sodium glyceryl trioleate sulfate; sodium hexadecyl diphenyl disulfonate; sodium hexadecyl diphenyloxide disulfonate; sodium hexyl diphenyloxide disulfonate; sodium isethionate; sodium isodecyl sulfate; sodium isooctyl sulfate; sodium isostearoyl lactylate; sodium isothdeceth-15 sulfate; sodium lactate; sodium lauramido DEA-sulfosuccinate; sodium laureth phosphate; sodium laureth sulfate; sodium laureth sulfosuccinate; sodium laureth-10 phosphate; sodium laureth-11 carboxylate; sodium laureth-12 sulfate; sodium laureth-13 acetate; sodium laureth-13 carboxylate; sodium laureth-3 carboxylate; sodium laureth-4 carboxylate; sodium laureth-4 phosphate; sodium laureth-6 carboxylate; sodium laureth-7 carboxylate; sodium laureth-7 sulfate; sodium laureth-8 sulfate; sodium lauroyl glutamate; sodium lauroyl lactylate; sodium lauroyl lactylate; sodium lauroyl methylaminopropionate; sodium lauroyl sarcosinate; sodium lauryl phosphate; sodium lauryl sulfate; sodium lauryl sulfoacetate; sodium lignate; sodium lignosulfonate; sodium methallyl sulfonate; sodium methyl lauroyl taurate; sodium methyl myristoyl taurate; sodium methyl oleoyl taurate; sodium methyl palmitoyl taurate; sodium methyl stearoyl taurate; sodium methylnaphthalenesulfonate; sodium m- nitrobenzenesulfonate; sodium myreth sulfate; sodium myristoyl glutamate; sodium myristoyl sarcosinate; sodium myristyl sulfate; sodium nonoxynol sulfate; sodium nonoxynol-10 sulfate; sodium nonoxynol-10 sulfosuccinate; sodium nonoxynol-15 sulfate; sodium nonoxynol-4 sulfate; sodium nonoxynol-5 sulfate; sodium nonoxynol-6 phosphate; sodium nonoxynol-6 sulfate; sodium nonoxynol-8 sulfate; sodium nonoxynol-9 phosphate; sodium nonoxynol-9 sulfate; sodium octoxynol-2 ethane sulfonate; sodium octoxynol-3 sulfate; sodium octyl sulfate; sodium octylphenoxyethoxyethyl sulfonate; sodium oleic sulfate; sodium oleth-7 phosphate; sodium oleyl phosphate; sodium oleyl sulfate; sodium oleyl sulfosuccinamate; sodium palmitoyl sarcosinate; sodium phenyl sulfonate; sodium propyl oleate sulfate; sodium stearoyl lactylate; sodium stearyl sulfosuccinamate; sodium trideceth sulfate; sodium trideceth-3 carboxylate; sodium thdeceth-6 carboxylate; sodium trideceth-7 carboxylate; sodium tridecyl sulfate; sodium thdecylbenzene sulfonate; sodium xylenesulfonate; stearoyl sarcosine; TEA- lauroyl glutamate; TEA-lauryl sulfate; tetrasodium dicarboxyethyl stearyl sulfosuccinamate; TIPA-laureth sulfate; triceteareth-4 phosphate; triceteth-5 phosphate; thdeceth-2 phosphate; trideceth-3 phosphate; thdeceth-5 phosphate; tridecyl phosphate; and trilaureth-4 phosphate; trioctyl phosphate.
In yet another embodiment, the anionic detergent is selected from the group consisting of glycolic acid ethoxylate octyl ether; glycolic acid ethoxylate oleyl ether; glycolic acid ethoxylate lauryl ether; poly(ethylene glycol) 4-nonylphenyl 3- sulfopropyl ether potassium salt; glycolic acid ethoxylate 4-tert-butylphenyl ether; glycolic acid ethoxylate oleyl ether; glycolic acid ethoxylate oleyl ether; poly(ethylene glycol) n-alkyl 3-sulfopropyl ether potassium salt; glycolic acid ethoxylate 4-nonylphenyl ether; poly(ethylene glycol) n-alkyl 3-sulfopropyl ether potassium salt; sodium cholate hydrate; sodium deoxycholate; sodium taurodeoxycholate hydrate; sodium taurocholate; sodium cholate hydrate; sodium deoxycholate; sodium taurodeoxycholate hydate; sodium taurocholate; glycolic acid ethoxylate octyl ether; glycolic acid ethoxylate oleyl ether; glycolic acid ethoxylate lauryl ether; poly(ethylene glycol)4-nonylphenyl 3- sulfopropyl ether potassium salt; glycolic acid ethoxylate 4-te/f-butylphenyl ether; glycolic acid ethoxylate oleyl ether; glycolic acid ethoxylate oleyl ether; poly(ethylene glycol) n-alkyl 3-sulfopropyl ether potassium salt; glycolic acid ethoxylate 4- nonylphenyl ether; and poly(ethylene glycol) n-alkyl 3-sulfopropyl ether potassium salt.
In an exemplary embodiment, the anionic detergent is selected from the group consisting of poly(ethylene glycol)4-nonphenyl 3-sulfopropyl ether potassium salt; poly(ethylene glycol) monolaurate, carrageenan lambda; polyoxyethylene(150)dinonylphenyl ether polyoxyethylene (Igepal<(R)> DM- 970); and nonyl nonoxynol-15 phosphate (Rhodafac RM710).
In a preferred embodiment, the anionic detergent is a salt of dodecyl sulfate, such as sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate. Most preferably, the anionic detergent is SDS.
In another embodiment, the the anionic detergent is not a salt of dodecyl sulfate, such as sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate.
The buffering agent may be any agent capable of buffering the pH to the desired value.
In one embodiment, the buffering agent is selected from the group consisting of sodium cyclohexane diaminetetraacetate (CDTA), N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid (BES), A- (2-Hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES), acetic acid or acetate (e.g. sodium acetate), citric acid or citrate, malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric acid, maleic acid, cacodylic acid, [beta][beta]'-Dimethylglutaric acid, carbonic acid or carbonate, 5(4)-Hydroxymethylimidazole, glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic acid, phosphoric acid or phosphate, sodium acetate, 2:4:6- collidine, 5(4)- methylimidazole, N-ethylmorpholine, triethanolamine, diethylbarbituric acid, tris(hydroxymethyl)aminomethane (Tris), 3-(N- Morpholmo)propanesulfonic acid; A- morpholinepropanesulfonic acid (MOPS), 2- morpholinoethanesulfonic acid (MES), piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES), N-[tris(hydroxymethyl)methyl]-2- aminoethanesulfonic acid (TES), 4-(2- Hydroxyethyl)piperazine-l-propanesulfonic acid (EPPS), N-(2-acetamido)-2- aminoethanesulfonic acid (ACES), phosphate, carbonate, ethylenediamine, imidazole buffers or combinations thereof.
In another embodiment, the buffering agent has a pKa at 25 0C of between 4.0 and 8.0, more preferably between 4.5 and 7.5, most preferably between 4.75 and 7. In one embodiment, the composition may further include a chelating agent capable of chelating a divalent cation. Chelating agents may be included because divalent cations are required for optimal activity of many RNases. I.e. when chelating agents are included, many RNases will be less active. Thus, in one embodiment, the composition comprises chelating agents such as EDTA, Citrate or CDTA.
Experiments have shown that the composition without any chelating agents is capable of protecting RNA against degradation. Therefore, in another embodiment, the composition does not comprise a chelating agent such as EDTA, citrate or CDTA. This can be favorable e.g. where enzymatic digestion of the samples is required, examples of enzymes could be proteinase, lysozyme, Mutanolysin and Lysostaphin.
Urea, even in very low concentrations may in some embodiments protect RNA from degradation. Therefore, in yet another embodiment, the composition comprises urea. In this embodiment, the concentration of urea is preferably below IM. In other embodiments, the concentration is below 10OmM; 1OmM; 5 mM, 4 mM, 3 mM, 2 mM, ImM; 0,1 mM; 0,0ImM and 0,001 mM respectively .
The composition may also comprise a chaotropic agent for denaturation of proteins and other cellular components. Exemplary chaotropic agents are guanidine thiocyanate, sodium thiocyanate, guanidinium chloride, sodium iodide, potassium iodide and urea. Preferred concentration of chaotropic agents are more than 1 M, more than 2 M, more than 3 M and between IM and 5 M.
In alternative embodiments, the composition does not comprise chaotropic agents at a concentration above 100 mM; above 10 mM; 1 mM; 0,1 mM; 0,01 mM and 0,001 mM. In one embodiment, the composition does not comprise a chaotropic agent.
RNA storage solution
In yet another embodiment of the invention, the composition is a RNA storage solution used for improving storage stability of RNA. The RNA may be purified, i.e. substantially free of other macromolecules of the cell or the RNA may still be present in a cell or in a virus (as will be further outlined below).
Preferably, the RNA storage solution is a stock solution for dilution for between dilution between 1 and 20 times.
Thus, when the RNA has been purified and suspended in water to generate a RNA solution, 1 volume of a 10 X concentrated stock solution of the RNA storage solution may be added to 9 volumes of RNA solution. If the RNA has been precipitated and dried, a IX concentrated RNA storage solution may be used for resuspension and subsequent storage.
When the RNA is present in cells, e.g. bacteria, the RNA storage solution may be added to the bacteria containing sample (e.g. urine) or bacterial culture at appropriate amounts, where after the sample can be stored with reduced or no degradation of the RNA. Such sample may be stored at room temperature (between 18 and 28 0C), between 0 and 5 0C or below 0 0C. In one embodiment, the sample may be heated to more than 80 0C or more preferably more than 94 ° C degrees before storage.
Thus, the RNA storage solution makes handling of samples comprising bacteria for RNA purification much easier since using the RNA storage solution lessens the requirements for fast transportation to the laboratory for either purification or storage in the freezer and later purification.
The RNA storage solution may be provided as any appropriate stock solution. However, stock solutions that are intended for the following dilutions are preferred: 1, 2, 4, 5 and 10.
Suspension solution
In one embodiment, the composition is a suspension solution for suspending (dissolving) precipitated RNA or for suspending RNA containing cells or virus present in the sample. In this embodiment, the cells are typically single cell organisms such as yeast and bacteria. However, it may also be used for plants cells or tissue cells, e.g. when the tissue or plant has been grinded.
Cell lysis solution In another embodiment, the composition is a cell lysis solution for lysing RNA containing cells or virus present in the sample.
The cell lysis solution may also comprise a lytic enzyme to facilitate lysis. The lytic enzyme may be added to the cell lysis solution prior to use of the cell lysis solution. I.e. the lytic enzyme could be supplied in a separate vial for addition to the cell lysis solution.
Preferably, the lytic enzyme is a peptidoglucan degrading enzyme, e.g. lysozyme, mutanolysin (a N-Acetyl Muramidase that cleaves the N-acetylmuramyl-β(l-4)-N- acetylglucosamine linkage of the bacterial cell wall polymer peptidoglycan- polysaccharide), lysostaphin (a zinc endopeptidase that cleaves the polyglycine cross-links in the peptidoglycan layer of the cell wall), labiase (contains β-N- acetyl-D-glucosaminidase and lysozyme activity), achromopeptidase (a lysyl endopeptidase), or a combination peptidoglucan degrading enzyme. The lytic enzyme may also be a protein degrading enzyme, e.g. proteinase K. The lytic enzyme may also be fungi cell wall glucan degrading enzyme. When the lytic enzyme is a peptidoglucan degrading enzyme, the cell lysis solution is particular suited for lysis of bacteria.
However, the cells as mentioned in any of the previous embodiments may be any cells. In a preferred embodiment they are selected from the group consisting of bacteria, yeast, fungi, plant cells and mammalian cells.
The bacteria may be gram-negative such as e.g. E.coli, Salmonella typhimunium, Klebsiella pneumoniae, a gram positive such as e.g. Staphlococcus aureus, Staphyloccocus spp, Stapylococcus saprophytic, Enterococcus faecalis, , or a mycobacterium. In a preferred embodiment, the sample is urine collected from a mammal, preferably from a human. In this embodiment, the cells may be cells of the mammal to be used e.g. for diagnosis or detection. The cells may also be bacteria or fungi. The sample may also be or comprise cells from a tissue sample or other body fluids such as whole blood, cerebrospinal fluid, plasma, saliva, semen, serum or synovial fluid.
In one embodiment, the sample is heated to at least 50 ° C to facilitate cell lysis, preferably heating is done after the sample has been mixed with the composition.
When the cell lysis solution is used for lysis of bacteria, it preferably comprises anionic detergent at a concentration between 0,01% and 0,5% and a buffering agent keeping the pH between 4 - 7.
RNA or DNA precipitation solution
In yet another embodiment, the composition is a RNA or DNA precipitation solution. I.e. the composition enables DNA or RNA precipitation while protecting the RNA from degradation. Preferably, in this embodiment the composition comprises salt at concentration of at least 300 mM. In a preferred embodiment, 2 volumes ethanol or 1 volume isopropanol is added to facilitate precipitation.
For DNA precipitation, the salt is preferably sodium or potassium for DNA precipitation. Such composition can e.g. be used to precipitate DNA during phenol extraction. It can also be used to precipitate DNA using centrifugation. As will clear, precipitation of DNA is one step towards RNA purification.
For RNA precipitation, the salt is preferably lithium and the composition may be used during extraction or centrifugation as mentioned above for DNA precipitation. Method of storing and/ or preserving RNA
A second aspect of the invention is a method comprising contacting the sample with a composition as described in the first aspect of the invention. Preferred embodiments of the method are described in the first aspect. They are briefly mentioned in the following.
In a preferred embodiment, the method is a method of storing RNA in a sample.
In another preferred embodiment, the method is a method of precipitating RNA or DNA.
In another embodiment, the method is a method of suspending cells comprising RNA or suspending precipitated RNA.
In yet another preferred embodiment, the method is a method of lysing cells or virus containing RNA. In this embodiment, the composition is preferably a cell lysis solution as outlined in the first aspect.
The methods may further comprise a step of selected from the group consisting of: a. Phenol extraction and RNA precipitation b. Immobilization c. Differential centrifugation (cesium chloride) d. Capturing using a capture probe
The RNA may be further processed e.g. by any of steps a-d just mentioned before being employed in detection methods. Alternatively it may be used directly in detection methods, such as cDNA preparation, PCR or qPCR microarray analysis, northern blotting, dot blotting, luminex etc.
A more specific embodiment of the cell lysis method comprises a) Storing the sample in RNA storage solution described in the earlier section at room temperature; b) treating the sample with lytic enzymes (optional); c) add anionic detergent to at least 0.5%, d) or add other lytic agents; and optionally e) heating the sample to at least 5O0C for 5 - 10 min to facilitate cell lysis
In a preferred embodiment, the cells are gram positive bacteria, and step b is non-optional.
A third aspect of the invention is a kit comprising a solution selected from the group consisting of:
a. A cell suspension solution as described in the first aspect. b. A cell lysis solution as described in the first aspect. c. A RNA precipitation solution as described in the first aspect. d. A RNA storage buffer according as described in the first aspect. e. Instructions for use
Preferably, the kit of comprises a. A cell suspension solution as described in the first aspect. b. A cell lysis solution according as described in the first aspect. c. Instructions for use
More preferably, the kit comprises a. A cell suspension solution as described in the first aspect. b. A cell lysis solution according as described in the first aspect. c. A RNA storage buffer according as described in the first aspect. d. Instructions for use In another embodiment the kit comprises a. A cell suspension solution as described in the first aspect. b. A cell lysis solution according as described in the first aspect. c. A RNA precipitation solution as described in the first aspect. d. A RNA storage buffer as described in the first aspect. e. Instructions for use
Exam ples
Exam ple 1 : I nhibition of H um an Urine RNase by low concentrations of SDS at different pH
Urine sam ple : Urine specimens from hospital patients were tested for bacterial growth at Clinical Microbiological laboratory, Hvidovre Hospital, Copenhagen. Fifty-three of the specimens lacking visible bacterial growth on nonselective growth medium were pooled, and the pH was determined to be 6.5, measured by a pH meter. The urine sample was stored in 3.5 ml aliquots at -2O0C,
Urine RNase activity was studied by the following procedure. The urine sample was buffered with either Sodium Phosphate or Sodium Acetate buffer to pH range 5.2 - 7.0, and SDS was added to a range of 0 - 0.1%. The final samples were of 12 μl volume and containing 58% of urine, 42 mM of Sodium Phosphate buffer or 42 mM Sodium Acetate buffer, 2 μg of purified E. coli total RNA (Ambion). The samples were incubated at room temperature for 15 min. Reactions were stopped by adding 3 μl of 10% SDS to each sample, and RNA degradation was analysed by electrophoresis on a 1.3% agarose gel in TAE runing buffer.
As shown in Fig. 1, when SDS was absent during the reaction time at room temperature, no rRNA bands were observed, whereas 0.05% and 0.1% SDS provided substantial protection in the entire pH range tested. At acidic pHs (e.g. pH 5.2, pH 5.7), SDS at as low as 0,025%, protected 16S and 23S rRNA from being degraded
Exam ple 2 : Preservation of rRNA in gram -negative bacteria in buffered human urine
E. coli culture : E. coli strain ATCC 25922 was cultured by the following procedure. Seven ml serum broth was inoculated with frozen £ coli stock and incubated at 370C overnight without agitation. One ml of the overnight culture was transferred to 9 ml fresh serum broth and subsequently incubated at 370C for 31/2 hr without agitation. £ coli cells were harvested in 1 ml aliquots by centrifuging at 9000 rpm for 2 min; the supernatant was discarded and the cell pellets were stored at -2O0C.
H uman urine sam ple containing E. coli. The urine sample (described in Example 1) was buffered with either Sodium Phosphate or Sodium Acetate buffer to pH 4.6 to 7.0; SDS was added to a range of 0 to 0.24%. £ coli was spiked into the buffered urine to approximately 5x 108 CFU/ml. The final samples were of 105 μl volume and containing 67% of urine, 48 mM of Sodium Phosphate buffer or 48 mM Sodium Acetate buffer. The samples were stored at room temperature. For analysis of RNA preservation, 10 μl of each sample was lysed by adding 2 μl of 10% SDS, and the preservation of 16S and 23S rRNA was analysed by electrophoresis on a 1.3% agarose gel in TAE runing buffer
As shown in Fig. 2, at pH 4.6, pH 5.2, pH 5.7, 16s/26S rRNA were preserved even after 10 days storage at room temperature.
Exam ple 3 : Preservation of RNA in gram-positive bacteria in buffered human urine containing SDS
Gram positive bacterial culture: Staphylococcus aureus (ATCC 29212), was cultured according to the procedure similar to that of E. coli culture described in Example 1. The bacterial cells were harvested in 1 ml aliquots and the cell pellets were stored at -2O0C as described in example 1. Human urine sample was buffered with sodium acetate (final concentration 48 mM) to pH 5.7, and spiked with Staphylococcus aureus. SDS was added to 0.12%. The final sample contains 71%, 38%, 19%, 9.5% , 4.8% or 0% urine (Fig. 3).
The samples were stored at room temperature for 4 days. For the lysis of gram positive bacteria, the samples were treated with a mixture of Hen Egg lysozyme (Fluka), Lysostaphin (Sigma), and mutanolysin (Sigma), in a final volume of 29 ul containing 0-62% urine, 0.1% SDS, 6 U mutanolysin, 6 U lysostaphin, 0.15 mg lysozyme, 43 mM Sodium Acetate buffer, with a final pH of 5.7. The samples were incubated at room temp for 30 min, and then SDS was added to 2%. Five μl of each lysates were analysed, by electrophoresis on a 1.3% agarose gel in TAE runing buffer.
Exam ple 4 : Detection of bacterial rRNA by capture assay
E. Co Ii lysate in urine
The urine sample (described in Example 1) was buffered with Sodium Acetate buffer to pH 5.0; SDS was added to 2%. £ coli was spiked into the buffered urine to approximately 5x 108 CFU/ml. The final samples were of 140 μl volume and containing 7% of urine, 45 mM Sodium Acetate buffer. The above mixture was incubated at room temperature for 15 min, and then incubated at 950C for 5 min. This total lysate was diluted in a 4-fold serial dilution in H2O, and used directly in the capture assays below.
Capture assay
The amine modified capture probe (EC0797:
CATCGTTTACGGCGTGGACTACCAGGG) was coupled to MagPlexTM-C Magnetic Carboxylated Microspheres (MC10015-04, Luminex Corporation, USA) by the carbodiimide coupling method provided by the manufacture. The capture assays were performed in a 96-well microwell plate, and each assay contains 5ul (contains approximate 2500 beads) of the beads carrying capture probe, 10 ul of rRNA sample (see the scheme in Table 1), 60 ul hybridization buffer (10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, IM NaCI), 25 ul of biotin labeled detection probe (lOuM ) (EC0766: CATCGTTTACGGCGTGGACTACCAGGG). The plate was incubated at 650C for 25 min, and then the beads were washed 3 times with 100 ul Washing buffer (10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, 250 mM NaCI). For detection assay, 100 ul of the Reporter Mix was added to each sample well (the detection mix contains 2.5 μg/ml of Strptavidin-R-PhycoErythrin (Invitrogen), 100 μg/ml BSA, 10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, and 250 mM NaCI), and subsequently incubated for 15 min at 250C. The beads were washed 3 times with 100 ul Washing buffer ((10 mM Sodium Phosphate Buffer (pH 7.4), 1 mM EDTA, 250 mM NaCI), and then resuspended in 100 μl of 0.5xTMAC buffer. Eighty μl_ of the beads were analysed on the Luminex analyzer, StarStation 3.0, according to the system manual (results are shown in Table IB). Ribosomal RNA from E. coli MRE600 (Roche Applied Science, cat. Nr. 10206938001, 4 μg/μl) was used as control rRNA.
Table IA: Scheme for capture assay
Figure imgf000020_0001
Table IB: Results of Capture assay given as MFI (Median fluorescence intensity)
Figure imgf000021_0001
Exam ple 5 : Com patibility of the lysis procedure w ith dow nstream nucleic acids purification procedure
Human urine sample was buffered with Sodium Acetate buffer to pH 5.2. The final samples were of 100 μl volume and containing 70% of urine, 50 mM Sodium Acetate buffer and 0,0.03%, 0.06%, 0.125%, or 0.25% SDS. One tube of the E.coli pellet from example 2 was resuspended in 65 μl of sterile H2O, and 5μl of the cell suspension was added to each of the samples above. The samples were stored at room temperature for 8 days.
At day 8, 30 μl of each of the samples above was lysed by adding 3.3 μl 20% SDS (i.e. final cone. 2%).. Nucleic acids were purified by the geneMAg-RNA/DNA kit according to manufacturer's instruction, except that smaller volumes of the magnetic beads and lysis/binding buffer were used (25μl of magnetic beads, and 500 μl of the buffer). The nucleic acids were eluted in 50μl of DEPC H2O; 15 μl of each preparation were analysed by electrophoresis on a 1.3% agarose gel in TAE runing buffer (results are shown in Fig.4)

Claims

Claims
1) Use of a composition for protecting RNA against degradation wherein said composition comprises
a. An anionic detergent at a concentration of between 0,01% and
0,5%, when the composition is mixed with a sample b. A buffering agent keeping the pH below 7, when the composition is mixed with the sample.
2) The use according to claim 1, wherein the pH is between 4,5 and 6.
3) The use according to any of the preceding claims , wherein the anionic detergent is selected from the group consisting of alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, alpha sulfonyl fatty acids, alkyl phosphates, dioctyl sulfosuccinate, isethionates, alkyl ether sulfates and methyl sarcosines
4) The use according to any of the preceding claims, wherein the alkyl sulfate is sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate.
5) The use according to any of the preceding claims, wherein the buffering agent is selected from the group consisting of sodium cyclohexane diaminetetraacetate (CDTA), N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid (BES), A- (2-Hydroxyethyl)piperazine-l- ethanesulfonic acid (HEPES), acetic acid or acetate (e.g. sodium acetate), citric acid or citrate, malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric acid, maleic acid, cacodylic acid, [ beta ][ beta ]'- Dimethylglutaric acid, carbonic acid or carbonate, 5(4)- Hydroxymethylimidazole, glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic acid, phosphoric acid or phosphate, sodium acetate,
2:4:6-collidine, 5(4)- methylimidazole, N-ethylmorpholine, triethanolamine, diethylbarbituric acid, tris(hydroxymethyl)aminomethane (Tris), 3-(N-Morpholmo)propanesulfonic acid; A- morpholinepropanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES), N- [tris(hydroxymethyl)methyl]-2- aminoethanesulfonic acid (TES), 4-(2- Hydroxyethyl)piperazine-l-propanesulfonic acid (EPPS), N-(2-acetamido)- 2-aminoethanesulfonic acid (ACES), phosphate, carbonate, ethylenediamine, imidazole buffers or combinations thereof.
6) The use according to any of the preceding claims, wherein the composition does not comprise chelating agents capable of chelating a divalent cation.
7) The composition according to any of the preceding claims, wherein the composition comprises urea.
8) The composition according to any of claims 1-6, wherein the composition does not comprise a chaotropic agent.
9) The use according to any of the preceding claims, wherein the composition is a suspension solution for suspending RNA containing cells present in the sample or for suspending precipitated RNA.
10) The use according to any of the preceding claims, wherein the composition is a cell lysis solution for lysing RNA containing cells present in the sample.
11) The use according to claim 10, wherein the cell lysis solution also comprises a lytic enzyme.
12) The use according to claim 11, wherein the lytic enzyme is added to the cell lysis solution prior to use of the cell lysis solution.
13) The use according to any of the preceding claims, wherein the cells are selected from the group consisting of bacteria, yeast, fungi, plant cells and mammalian cells
14) The use according to claim 13, wherein the bacteria is gram positive bacteria selected from the group consisting of Staphlococcus aureus, Staphyloccocus spp, Stapylococcus saprophytic, Enter ococcus faecal is or a mycobacterium 15) The use according to any of the preceding claims, wherein the sample comprises cells from a urine sample.
16) The use according to any of the preceding claims, wherein the composition is a RNA storage solution used for improving storage stability of RNA.
17) The use according to claim 16, wherein the RNA storage solution is a stock solution for dilution between 1 and 20 times.
18) A method of storing RNA in a sample comprising contacting the sample with a composition as described in any of claims 1-17.
19) The method of claim 18, wherein the RNA in the sample is present within cells or virus.
20) The method of claim 18, wherein the RNA is purified.
21) A method of extracting RNA from a biological sample comprising contacting the sample with the composition as described in any of claims
1-17.
22) The method of claim 21, wherein the composition is the cell lysis solution of any of claims 10-15 and the biological sample comprises bacterial cells.
23) The method of any of claims 18-22 further comprising a step of RNA purification selected from a. Phenol extraction and RNA precipitation b. Immobilization c. Differential centrifugation
24) The method of claim 23, wherein the RNA is used directly in a method selected from the group consisting of microarray analysis, cDNA preparation, qPCR and northern blot. 25) A kit for RNA extraction comprising a solution selected from the group consisting of:
a. A suspension solution as described in claim 9 b. A cell lysis solution as described in any of claims 10-15 c. A RNA storage buffer as described in any of claims 16-17 d. Instructions for use
26) The kit of claim 25 comprising a. A suspension solution as described in claim 9 b. A cell lysis solution as described in any of claims 10-15 c. Instructions for use
27) The kit of claim 25 comprising a. A suspension solution as described in claim 9 b. A cell lysis solution as described in any of claims 10-15 c. A RNA storage buffer as described in any of claims 16-17 d. Instructions for use
PCT/DK2010/050018 2009-01-26 2010-01-26 Methods and uses for rna extract and storage WO2010083844A1 (en)

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JP2014097051A (en) * 2012-08-10 2014-05-29 Tosoh Corp Bacteriolytic reagent for acid-fast bacteria and method of detecting acid-fast bacteria with the same
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