EP2231853A2 - Procédé et trousse de travail pour la conservation d'échantillons et pour la désagrégation des cellules, avant l'extraction des acides nucléiques - Google Patents

Procédé et trousse de travail pour la conservation d'échantillons et pour la désagrégation des cellules, avant l'extraction des acides nucléiques

Info

Publication number
EP2231853A2
EP2231853A2 EP08865515A EP08865515A EP2231853A2 EP 2231853 A2 EP2231853 A2 EP 2231853A2 EP 08865515 A EP08865515 A EP 08865515A EP 08865515 A EP08865515 A EP 08865515A EP 2231853 A2 EP2231853 A2 EP 2231853A2
Authority
EP
European Patent Office
Prior art keywords
organic liquid
water
nucleic acids
cells
extraction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08865515A
Other languages
German (de)
English (en)
Inventor
Rudolf Ehwald
Dietmar Lerche
Holger Woehlecke
Christina Lerche
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dr Lerche KG
Original Assignee
Dr Lerche KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dr Lerche KG filed Critical Dr Lerche KG
Publication of EP2231853A2 publication Critical patent/EP2231853A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/063—Lysis of microorganisms of yeast

Definitions

  • the invention relates to a method for sample preservation and cell disruption, which can be used to prepare the extraction of nucleic acids from living cells, cell aggregates and tissue samples, as well as a work set for performing the method.
  • the method comprises introducing living cells, cell aggregates or tissue samples whose nucleic acids are to be extracted into an excessively water-miscible and volatile organic liquid.
  • the saturation of the sample with the organic liquid has an advantageous effect on the extraction of the nucleic acids, which then takes place in an aqueous medium. By almost completely removing the water from the cells, the mechanical cell disruption is facilitated. In the organic liquid, the biomembranes are dissolved.
  • TRNAs and other RNA fractions with a low degree of polymerization which can permeate through the cell wall, can be selectively extracted without mechanical disruption.
  • the cells, cell aggregates or tissue samples Prior to the extraction of high molecular weight nucleic acids, the cells, cell aggregates or tissue samples are digested in the organic liquid by means of mechanical impulses. Since the nucleases are inactive in the dewatered organic liquid, the biological samples or the homogenates prepared from them can be stored in the organic liquid at room temperature for as long as desired. After incubation in the largely anhydrous organic liquid, the sample-specific nucleases are largely denatured.
  • An advantageous working utensil according to the invention contains an organic / aqueous mixed phase with denaturing properties and, for each sample, a dewatering shaped body adapted to the size of the preserving vessel and a mixture of a few large and numerous small digestion bodies adapted to the size of the preserving vessel for digestion in a nonaqueous medium.
  • An essential advantage of the invention is that can be dispensed with cooling and strong mechanical impulses in the disruption of the cells and in the extraction and that the extraction of DNA can be made time independent of the disruption of the cells.
  • nucleic acids In the extraction of nucleic acids from living biological material, it is necessary for numerous molecular biology applications, the enzymatic degradation of this To prevent macromolecules in the production of the cell-free extract.
  • bacteria, fungi and plant cells must be disrupted mechanically or enzymatically because the cell walls are impermeable to high molecular weight nucleic acids.
  • the most common method currently used is mechanical disruption of cells frozen in liquid nitrogen, cell aggregates or tissue samples (eg, tissue particles, tissue sections) and their subsequent transfer to an aqueous extraction or lysis buffer.
  • the energy input necessary for the mechanical digestion causes a risk of local thawing and the possible reaction of nucleases.
  • nucleases present in the lysosomes, vacuoles and cell walls upon inactivation of cells are rapidly inactivated. This is achieved imperfectly in biological samples with high endogenous RNAse activity by the usual combination of denaturing additives to the extractant, removal of calcium ions by chelators, low temperatures and short extraction time for biological samples with high endogenous nuclease activity and with reduced yield.
  • the patent US 6204375 describes a method and a means for preserving RNA in biological samples. It is based on the denaturing, dehydrating and the endogenous nucleic acids precipitating effect concentrated salt solutions and allows for longer storage of the preserved samples, the subsequent extraction of high molecular weight RNA in good quality.
  • the high salt concentration in the preservation solution may be detrimental to the following studies.
  • the problems associated with cell disruption are retained.
  • the task derives from preserving high molecular weight DNA and RNA of living cells, cell aggregates and tissue samples at normal temperature and to develop a method for the mechanical digestion of plant cells, fungi and bacteria, while largely avoiding enzymatic or mechanical depolymerization of the nucleic acids Normal temperature can be performed.
  • the invention is based on the finding that very resistant yeast and bacterial cells or plant tissue with low mechanical performance can be completely broken up in a short time against mechanical disruption, after they are dissolved in an organic water-miscible volatile liquid, for example acetone or ethanol, were largely drained.
  • an organic water-miscible volatile liquid for example acetone or ethanol
  • a substantially anhydrous organic liquid is to be understood that the water content of the organic liquid is smaller than the water content of the azeotropic mixture of the liquid with water.
  • the maximum water content is 4%, since 96% ethanol at 78.4 0 C azeotropically with a water content of 4% can be distilled.
  • a generator of mechanical pulses in a volume of liquid e.g. a shaker, a stirrer, a manually operated stirring rod, an ultrasonic source or the like required.
  • Essential to the invention is that the mechanical impulses act on a biological material that is in a largely water-free, water-miscible and volatile liquid.
  • An advantageous variant of the method according to the invention is to carry out the cell disruption in the preservation and digestion vessel used for dehydration in the organic liquid with the aid of suitable digestion particles.
  • Preference is given to using fine pulping particles of glass with a diameter of less than 0.5 mm with a volume fraction of 5 to 30% and some larger and heavier pulping particles of glass or steel having a diameter of 2 to 5 mm.
  • the larger digestion bodies increase the flow velocity gradients in the digestion vessel due to their inertia.
  • a high concentration of fine disruption particles increases the frequency of the cell-wall-breaking mechanical impulses, which act on the dehydrated cells, cell aggregates or tissue samples.
  • the digestion particles are used in an approximately anhydrous medium of the organic liquid in a similar manner as is usual for cell disruption in an aqueous medium.
  • the use according to the invention of digestion particles in an approximately water-free liquid medium has the advantage that a loss-free and complete disruption of the cells with low energy input is possible. As the cell walls lose their elasticity in the absence of water, cells or tissue particles are more likely to burst under the influence of shear when dehydrated.
  • the device is based on the fact that the mechanical impulses and shearing forces required to produce a homogenate of mechanically disrupted cells, cell aggregates or tissue samples act on the cells or tissue samples in the substantial absence of the swelling agent.
  • the high molecular weight nucleic acids are present in highly condensed form and are thereby protected from degradation by mechanical shear.
  • the mechanical disruption of cells and tissues in a well dehydrated organic liquid therefore does not result in degradation of the DNA by shear.
  • the water which has passed into the organic liquid can be removed in the simplest case by repeated renewal of the organic liquid, a water-containing organic medium being exchanged for anhydrous.
  • the biological material used as is typical for tissue samples of plants and fungi, has high endogenous activities of nucleases, phenol oxidases and other oxidizing or radical-releasing enzymes, by the gradual increase in the concentration of the organic liquid into the cells Nucleic acid degradation takes place.
  • the extractability of the nucleic acids can be impaired by formation of polyphenols and oxidative protein crosslinking.
  • the enzymatic processes associated therewith can be suppressed according to the invention by introducing the living tissue samples into a mixture of the organic liquid and a buffer of low pH (pH 2-4), whose proportion in the total volume is 5 to 30%. This mixture is replaced after a short exposure time (preferably 30 min to 2 h) against a largely anhydrous organic liquid.
  • a suitable reducing agent eg, sodium sulfite, sodium dithionite, mercaptoethanol, and the like may be added to the mixture.
  • the tissue samples first in an organic liquid with a water content of about 20% and has to replace this liquid after a short time against the largely anhydrous organic liquid has another advantage.
  • the vessel used for the sample preservation with the organic volatile liquid may also be added to a suitable for their dewatering solid.
  • a suitable for their dewatering solid particularly porous zeolite (molecular sieve) with a pore size of 0.3 nm, which is known under the name Molsieb used for drying alcohol and other organic liquids.
  • porous zeolite molecular sieve
  • anhydrous crystals of sodium sulfate, magnesium sulfate or the like are also be added in sufficient quantity.
  • the product of the bulk of the dehydrating agent and its water-binding capacity relative to the mass should exceed the water content of the sample. If the dehydrating agent is used in sufficient quantity, can be dispensed with the multiple replacement of the organic liquid.
  • An important advantage of the method and apparatus of the invention is that organic, water-miscible volatile liquids such as acetone, dioxane, methanol, ethanol and iso-propanol destroy the lipid membranes and rapidly invade cells and small tissue sections, causing them dehydrated and the nucleases and the oxidation-active enzymes are deactivated.
  • organic, water-miscible volatile liquids such as acetone, dioxane, methanol, ethanol and iso-propanol destroy the lipid membranes and rapidly invade cells and small tissue sections, causing them dehydrated and the nucleases and the oxidation-active enzymes are deactivated.
  • the said enzymes are also irreversibly denatured.
  • the saturated with the organic liquid tissue samples or cells or their homogenates, which were prepared by mechanical disruption of cells or tissue particles in the anhydrous organic liquid can be stored for any length of time without replacement of the organic liquid medium against the aqueous extraction buffer without the risk of enzymatic changes at room temperature become. Since the cell's own nucleases and possibly also nucleases that have been contaminated by the sample are denatured by extensive dehydration in the anhydrous, water-miscible organic liquids, the sample is usually free of active nucleases after cell disruption. Therefore, the subsequent extraction of the nucleic acids in an aqueous extractant without nuclease inhibitors can be carried out at room temperature.
  • the organic liquid can be easily separated from particulate cell residues containing the nucleic acids in undissolved form by centrifugation. Because of the large difference in density and the low solvation of the hydrophilic swelling substances in the particulate cell residues, the dewatered cell debris in the organic liquid sediments completely and in a short time at low speed. The volatility of the organic liquid then allows the complete removal of the organic liquid by evaporation. This process can be rationalized. For example, if 2 ml polypropylene sample tubes (Eppendorf tubes) are used as preservation and sample digestion vessels, after decanting or aspirating the majority of the supernatant organic liquid, the residual liquid is obtained by briefly incubating the vessel in vacuo at room temperature or heating in a commercial Heating block completely removed.
  • Eppendorf tubes polypropylene sample tubes
  • insoluble fine disruption particles and cell wall fragments are removed from the aqueous extraction fluid by microfluidization, centrifugation or simple sedimentation in order to obtain a clear aqueous crude extract.
  • a separate step for the separation of particulate residues from the aqueous extract can be dispensed with if a chromatographic separation bed is used for the subsequent isolation of the nucleic acids. In this case, it is advantageous to cover the chromatographic separation bed in the used disposable column with a microfilter and to allow the extract with the insoluble constituents to flow into the separation bed via this microfilter.
  • an inventive working utensils can be used. It comprises a suitable organic liquid and for each sample a solid nearly anhydrous dehydrating agent which is suitable in the organic Liquid to bind dissolved water. It is particularly advantageous if the dehydrating agent is present as a solid body and is adapted in its shape and mass to the vessel so that it can be conveniently removed again after the dehydration of the organic liquid. In order to prevent the dry dehydrating agent from absorbing water vapor from the air, the dewatering body adapted to the vessel is packed in a water vapor-tight manner. After dehydration of the sample, the dehydrating agent is removed again from the preservative vessel. Subsequently, the digestion particles are added to the organic liquid with the sample. Now, the preservation vessel can be used for cell disruption by shaking the sample with the disruption particles.
  • the working utensil according to the invention for each sample contains an adapted to the vessel amount of digestion particles, preferably in the form of a mixture of heavy particles that generate strong flow in the organic liquid during shaking and smaller particles containing the Accelerate disruption of small cells and tissue by ensuring a high pulse density.
  • digestion particles preferably in the form of a mixture of heavy particles that generate strong flow in the organic liquid during shaking and smaller particles containing the Accelerate disruption of small cells and tissue by ensuring a high pulse density.
  • the vessels were occasionally or constantly shaken. After 24 h or 8 weeks, cell disruption was performed.
  • the cell suspension was separated by decantation from the dehydrating agent and transferred to a sealable sample digestion vessel containing 200 mg of fine glass beads with a diameter of 1 to 10 microns (Spheriglas Potters Europe, Suffolk, UK) and 10 glass beads with a diameter of about 2 mm contained.
  • the digestion vessels (Eppendorf tubes) were shaken in a horizontal position on a simple commercial small shaker for mixing liquids (vortex mixer). The light microscopic evaluation revealed that the cells were almost completely digested after 30 minutes. In contrast, the yeast cells in control samples that were not dried with sodium sulfate were only partially disrupted.
  • the residue was shaken with 0.4 ml of an aqueous extraction buffer (10 mM Tris, 10 mM EDTA, pH 8, 1.4% sodium dodecyl sulfate) on the mixer for 10 minutes and then incubated at 37 ° C.
  • an aqueous extraction buffer (10 mM Tris, 10 mM EDTA, pH 8, 1.4% sodium dodecyl sulfate) on the mixer for 10 minutes and then incubated at 37 ° C.
  • 50 ⁇ l of these dispersions were applied after different times to a column for the isolation of nucleic acids according to DE 199 02 724 and DE 102004030878.
  • the columns contained over the sporopollenin microcapsule separation bed (1 ml) filter layer (3 mm) of fine particles (5-10 ⁇ m). Elution was with Na-EDTA (10mM, pH 8).
  • RNA molecules of different molecular mass were also detectable.
  • the duration of the preservation period 24 h or 8 weeks had no influence on the sharpness and position of the bands of the electropherogram. It is noteworthy that even with long extraction times (up to 24 h) and the high temperature during the Extraction (37 ° C) the high molecular weight RNA and DNA remained stable. Electrophoretically, no degradation of the nucleic acids in the extraction buffer was found. It follows that the preservation and digestion process of the invention irreversibly denatures the endogenous nucleases.
  • the extraction can be carried out in suitable buffers without risk of undesired enzymatic degradation at room temperature and also at elevated temperature over a longer period of time. Therefore, high yields of DNA and high molecular weight RNA can be achieved.
  • Ethanol was used as the organic liquid.
  • a yeast suspension was prepared by mixing with water in a ratio of 1/1. Samples of 50 ⁇ l of this suspension were added to 2 ml Eppendorf tubes with 1 ml of 96% ethanol. A portion of the samples (variant A) were shaken at room temperature overnight and then collected by centrifugation. In another part of the samples (variant B), the yeasts were collected by centrifugation after a shaking time of 4 h, the pellet was admixed with 1 ml of absolute ethanol and further shaken overnight. In another part of the samples (variant C), the water-containing ethanol was also exchanged for absolute ethanol.
  • a water-absorbent molding was introduced in variant C for the complete removal of the water from the ethanol-saturated cells and removed again after a drainage time of 18 hours.
  • the water-absorbent molded articles used were prepared by adhering twenty 3 mm diameter zeolite beads 0.3 nm pore size to a 1.8 cm long and 4 mm wide cellulose strip, in a freeze dryer at a shelf temperature of 80 0 C in vacuum (0.07 mbar) completely dehydrated and packaged vapor-tight in an Eppendorf tube.
  • a digestion body mixture consisting of 10 glass beads with a diameter of 3 mm and 100 mg of glass beads with a diameter of 0.2 to 0.5 mm was added to the suspensions after a dehydration time of 18 h.
  • the tubes were placed on a vortexing shaker with a frequency of 50 s -1 and a shaking radius of 2.5 mm 30 shaken for a long time.
  • no glass beads were added and the tubes were also not shaken.
  • the extraction of the nucleic acids in the aqueous extraction buffer was carried out in the same manner in all variants at room temperature.
  • 0.1 ml of the ethanolic suspensions of cells or cell residues were removed and centrifuged in a 0.5 ml Eppendorfrschreibchen. The ethanol was decanted. The pellets were taken up in 0.1 ml of aqueous extraction buffer (1.4% SDS, 50 mM EDTA-Na, pH 7.4) and shaken on an overhead shaker for about 18 hours. The extracts were freed of particles by centrifugation. 10 ⁇ l of the centrifuged extracts were analyzed electrophoretically by gel electrophoresis in an agarose gel (1.3%).
  • Variants B and C revealed a sharp band of high molecular weight DNA and two sharp bands of rRNA (28 S and 18 S rRNA).
  • Low molecular weight RNA (mainly tRNA) about 80 nucleotides in size was also seen.
  • variant C complete dehydration of the ethanol by zeolite
  • the bands of high molecular weight RNA and DNA were more prominent than in variant B.
  • the extracts of the mechanically undigested ethanol treated yeast cells contained no detectable amounts of high molecular weight nucleic acids. However, they contained low molecular weight RNA (especially tRNA) in a comparable amount as the extracts of variants B and C.
  • variants A, B and C were centrifuged and taken up with aqueous extraction buffer containing methylene blue (concentration 0.2%) In the microscope, the disrupted cell wall envelopes can easily be distinguished from the strongly blue-colored denatured cells with an intact cell wall in this staining. While in variant A all cell walls were uninjured, in variant C all cells were open. In variant B, about 60% of the cells were disrupted.
  • RNA molecules of the size of the t-RNA could be removed from the intact cells with the help of the Accordingly, after the preservation of the nucleic acids according to the invention with a water-miscible organic liquid, the cell wall can be utilized as an ultrafilter for the selective aqueous extraction of low molecular weight RNA fractions Water-miscible organic liquid, allows the selective extraction of a low molecular weight RNA size fraction. This is very advantageous for the investigation of small RNA molecules, such as siRNA, miRNA and tnRNA.
  • tissue samples were completely homogenized. Apart from cell debris, only open cells were visible in the microscope. Smaller pieces of connected cells were confined to xylem vessels and small areas of the epidermis, all of whose cells were open. Comparative samples in which fresh tissue were shaken with equal intensity in water, were not or only very incompletely homogenized.
  • the good quality of the extracted high-molecular nucleic acids is shown by the finding that the rRNA band for the 28 S subunit was stronger than that of the small subunit. It was also detectable in the plant tissues that the inventive method ensures a high stability of the high molecular weight nucleic acids in the subsequent extraction with an aqueous extractant. The same result was obtained even when the ethanolic homogenates were stored for 4 weeks at room temperature.

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Abstract

L'invention concerne un procédé de conservation d'échantillons et de désagrégation de cellules, utilisable pour la préparation de l'extraction d'acides nucléiques à partir de cellules vivantes, de groupes cellulaires et d'échantillons de tissus. L'invention concerne en outre une trousse de travail pour la mise en oeuvre du procédé. Le procédé comprend l'introduction de cellules vivantes, de groupes cellulaires ou d'échantillons de tissus dont on doit extraire les acides nucléiques, dans un liquide organique en excès, miscible à l'eau et volatil. La saturation de l'échantillon par le liquide organique agit avantageusement sur l'extraction ultérieure, en milieu aqueux, des acides nucléiques. La désagrégation mécanique des cellules est facilitée par l'élimination quasi complète de l'eau des cellules. Les biomembranes sont dissoutes dans le liquide organique. Les fractions tARN et les autres fractions ARN à faible degré de polymérisation, qui peuvent pénétrer à travers la paroi cellulaire, peuvent être extraites sélectivement sans désagrégation mécanique. Avant l'extraction des acides nucléiques hautement moléculaires, les cellules, les groupes cellulaires ou les échantillons de tissus sont désagrégés dans le liquide organique à l'aide d'impulsions mécaniques. Du fait que dans le liquide organique débarrassé de l'eau, les nucléases sont inactives, les échantillons biologiques ou les homogénats qui en résultent dans le liquide organique sont conservés pendant une durée à volonté, à température ambiante. Après incubation dans le liquide organique en majeure partie exempt d'eau, les nucléases appartenant aux échantillons, sont dénaturées en majeure partie. L'invention concerne en outre une trousse de travail avantageuse contenant une phase mixte organique/aqueuse aux propriétés dénaturées, et un corps moulé de déshydratation adapté à la grandeur du récipient de conservation, ainsi qu'un mélange, adapté à la grandeur du récipient de conservation, de quelques gros corps de désagrégation et de nombreux petits corps de désagrégation, pour la désagrégation en milieu non aqueux. Un avantage essentiel de l'invention réside dans le fait qu'on peut supprimer le refroidissement et une forte impulsion mécanique lors de la désagrégation des cellules et lors de l'extraction, et que l'extraction de l'ADN peut se développer indépendamment, dans le temps, à partir de la désagrégation des cellules.
EP08865515A 2007-12-21 2008-12-22 Procédé et trousse de travail pour la conservation d'échantillons et pour la désagrégation des cellules, avant l'extraction des acides nucléiques Withdrawn EP2231853A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007063019A DE102007063019A1 (de) 2007-12-21 2007-12-21 Verfahren und Arbeitsbesteck zum Aufschluss von Zellen sowie zur Konservierung und Extraktion von Nukleinsäuren aus lebenden Zellen oder Geweben
PCT/EP2008/068210 WO2009080824A2 (fr) 2007-12-21 2008-12-22 Procédé et trousse de travail pour la conservation d'échantillons et pour la désagrégation des cellules, avant l'extraction des acides nucléiques

Publications (1)

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EP2231853A2 true EP2231853A2 (fr) 2010-09-29

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EP08865515A Withdrawn EP2231853A2 (fr) 2007-12-21 2008-12-22 Procédé et trousse de travail pour la conservation d'échantillons et pour la désagrégation des cellules, avant l'extraction des acides nucléiques

Country Status (4)

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US (1) US20110098462A1 (fr)
EP (1) EP2231853A2 (fr)
DE (1) DE102007063019A1 (fr)
WO (1) WO2009080824A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8927214B2 (en) 2012-04-18 2015-01-06 Syngenta Participations Ag Methods and compositions for dual extraction of protein and nucleic acid
CA2961613A1 (fr) 2014-09-17 2016-03-24 Hologic, Inc. Procede de lyse partielle et dosage
US11591591B2 (en) 2019-08-21 2023-02-28 New England Biolabs, Inc. Isolation of high molecular weight DNA using beads

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6204375B1 (en) * 1998-07-31 2001-03-20 Ambion, Inc. Methods and reagents for preserving RNA in cell and tissue samples

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6244170A (ja) * 1985-08-19 1987-02-26 Agency Of Ind Science & Technol モルテイエレラ属糸状菌体の超臨界流体による抽出方法
DE19902724A1 (de) 1999-01-19 2000-07-27 Dietmar Lerche Mikrokapseln aus Sporopollenin, Verfahren zu ihrer Herstellung und Anwendung
DE102004030878B4 (de) 2004-06-25 2008-05-08 Dr. Lerche Kg Verfahren zum selektiven Konzentrieren und Sammeln chromatographisch getrennter Stoffe und Sammelvorrichtung für die Flüssig-Chromatographie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6204375B1 (en) * 1998-07-31 2001-03-20 Ambion, Inc. Methods and reagents for preserving RNA in cell and tissue samples

Also Published As

Publication number Publication date
US20110098462A1 (en) 2011-04-28
WO2009080824A3 (fr) 2009-12-03
WO2009080824A2 (fr) 2009-07-02
DE102007063019A1 (de) 2009-07-02

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