WO2009040444A1

WO2009040444A1 - Rna isolation method

Info

Publication number: WO2009040444A1
Application number: PCT/EP2008/063039
Authority: WO
Inventors: Arne Deggerdal; Andrew Campbell; Veronica Boger
Original assignee: Mole Genetics As
Priority date: 2007-09-28
Filing date: 2008-09-29
Publication date: 2009-04-02
Also published as: GB0719022D0; GB201006994D0; GB2466419A; GB2466419B

Abstract

A method for isolating RNA from a biological sample, which comprises: (i) treating the sample with a lysis solution comprising a detergent at a pH of greater than 6 to form a lysate comprising an RNA-containing liquid phase; (ii) contacting the lysate with a first solid phase bearing a positive charge in the presence of a salt so as to precipitate DNA and protein from the RNA- containing liquid phase onto the first solid phase; (iii) separating the RNA-containing liquid phase from the solid phase; (iv) contacting the RNA-containing liquid phase with a non-ionic solid phase in the presence of a lower alcohol comprising ethanol or propanol to precipitate RNA onto the non-ionic solid phase to form an RNA-containing solid phase; (v) separating the RNA-containing solid phase from the liquid phase; and (vi) isolating RNA therefrom.

Description

ISOLATION METHOD

Field of the Invention

The present invention relates to a method for isolating RNA from a biological sample, kits therefore, to purification of the isolated RNA, to the use of automated magnetic separation apparatus in the method and provision to the apparatus of data comprising instructions for operating steps of the method.

This invention is of great importance in the fields of molecular biology, biochemistry, gene technology, medicine, veterinary medicine and all related fields.

Background of the Invention

Deoxyribonucleic acid (DNA) and RNA have core functions in the control of biological processes. In most living organisms, genetic information is stored as DNA, which is a stable, double-stranded polynucleotide. Expression of the genetic information stored in DNA is accomplished by transcription of RNA from a DNA template, followed by translation of the RNA-encoded information into protein.

Due to the structural similarity between DNA and RNA, previous RNA purification methods have often comprised isolating DNA and RNA together from biological sources. Typically these methods have utilized phenol or a mixture of phenol and chloroform in a manor that denatures and precipitates proteins while leaving nucleic acids in solution. These methods are hazardous, laborious and of limited utility for isolation of RNA from biological sources containing high amounts of ribonuclease (RNAse), an extremely stable enzyme that degrades RNA.

Alternative methods for isolating RNA from ribomiclease-rich tissues have been disclosed (e.g. Chirgwin et al., Biochemistry, 18: 5924-29 (1979)). These methods include the use of strong chaotrophic salts (guanidinium thiocyanate) and 2- mercaptoethanol. It is well known in the field that chaotrophic salts eliminate nucleolytic hydrolysis of RNA by ribonuclease. RNA isolated in this manner often requires extensive, manipulation to further purify the RNA from other cellular contaminants e.g. DNA, protein and other cellular materials. A simpler method for isolating both DNA and RNA from biological sources was disclosed by Boom et al., J. Clin. Micro., 28:495-503 (1990), U.S. Pat. No. 5,234,809. Ceils present in biological sources, such as serum or urine, were lysed by exposure to strong (greater than 5M) solutions of GuSCN in Tris HCl (pH 8.0), containing EDTA and Triton X-100. DNA and RNA were purified from the mixture of biological materials by incubation with diatomaceous earth or silica particles, which formed reversible complexes with the DNA and RNA.

Miller et al., Nucl. Acids Res 16:1215 (1988) disclosed a simple salting out procedure for the extraction of DNA. Cells are first lysed then the cellular proteins are salted out by dehydration and precipitation with a saturated salt solution. The DNA is subsequently precipitated out from the supernatant using alcohols.

Current methods for the specific isolation of RNA typically incorporate techniques to disrupt or lyse cells and protect RNA from degradation by endogenous RNases. Lysis liberates RNA along with DNA and protein from which the RNA must then be separated.

Methods have been disclosed that utilize a low pH step to separate RNA from protein and DNA by extraction with phenol/chloroform (D. M. Wallace, Meth. Enzym., 15, 33-41 (1987)). A one-step isolation of RNA by treating cells sequentially with 4 M guanidinium salt, sodium acetate (pH 4), phenol, and chloroform/isoamyl alcohol followed by precipitating RNA from the upper layer by the addition of alcohol has been disclosed by Chomczynski, (Anal. Biochem., 162, 156-159 (1987)). U.S. Pat. No. 4,843,155 incorporates a method in which a stable mixture of phenol and guanidinium salt at an acidic pH is added to the cells.

Another method for isolating nucleic acids is disclosed in U.S. Pat. No. 6,355,792. The method comprises acidifying a liquid sample with a buffer having a pH less than 6.5 and contacting the acidic solution with an inorganic oxide material having hydroxyl groups, separating the solid material with bound nucleic acids on it from the liquid, and eluting with alkaline solution having a pH between 7.5 and 11. WO00/66783 discloses a method of extracting nucleic acids from a sample in the absence of a lysis step by contacting a sample containing a nucleic acid at a pH of less than 7, with a water-soluble, weakly basic polymer to form a water-insoluble precipitate of the weakly basic polymer and the nucleic acids, separating the precipitate from the sample, and contacting the precipitate with a base to raise the solution pH to greater than 7, thereby releasing the nucleic acids from the weakly basic polymer.

U.S. Pat. No. 5,973,137 to Heath discloses a method for isolating substantially undegraded RNA from a biological sample by carefully treating (not mixing more than 3 times) the sample with a cell lysis reagent consisting of an anionic detergent, a chelating agent and a buffer solution having a pH less than 6. The role of the anionic detergent is said to lyse cells and/or solubilize proteins and lipids as well as to denature proteins. When used to isolate RNA from whole blood, red blood cells are first lysed with a reagent containing NH₄Cl, NaHCO₃ and EDTA, the white blood cells are separated and separately lysed in the presence of a protein-DNA precipitation reagent. The latter is typically a high concentration of a sodium or potassium salt such as acetate or chloride. As a final step, the supernatant containing RNA is precipitated by addition of a lower alcohol.

U.S. Pat. No. 5,973,138 discloses a method for the reversible binding of DNA and RNA to a suspension of paramagnetic particles in acidic solution. The paramagnetic particles disclosed therein are bare iron oxide, iron sulfide or iron chloride. The acidic solution is said to enhance the electropositive nature of the iron portion of the particles and thereby promote binding to the electronegative phosphate groups of the nucleic acids. U.S. Pat. No. 6,433,160 discloses a similar method wherein the acidic solution contains glycine HCl.

U.S. Pat. No. 6,737,235 to Cros et al., discloses a method for isolating nucleic acids using particles comprising or coated with a hydrophilic, cross-linked polyacrylamide polymer containing cationic groups. Cationic groups are formed by protonation at low pH of amine groups on the polymer. Nucleic acids are bound in a low ionic strength buffer at low pH and released in a higher ionic strength buffer. GB 2419594 Al discloses the stabilization of nucleic acids with amino surfactants and optionally with nonionic surfactants

Cationic detergents are preferred stabilizing agents in U.S. Pat. Nos. 6,602,718; 6,617,170; and 6,821,789; and US Patent Application Publ. 2005/0153292 disclose. The cationic detergent is said to preserve RNA and/or DNA by inhibiting or blocking gene induction or nucleic acid degradation. The gene induction blocking agent can comprise a stabilizing agent and an acidic substance. The latter agents lyse cells and cause precipitation of nucleic acids as a complex with the detergent.

Compositions and methods for stabilizing nucleic acids comprising alcohols and/or ketones in admixture with dimethyl sulfoxide have been disclosed (U.S. Pat. No. 6,916,608B2).

it has been considered that the stabilization of the RNA content of cells can be effected by adding a solution of a salt such as ammonium sulfate at a pH between 4 and 8 (U.S. Pat. Nos. 6,204,375 and 6,528,641). The salt solution is said to permeate cells and causes precipitation of RNA along with cellular protein and renders the RNA inaccessible to nucleases which might otherwise degrade it.

From the abovementioned, it will be appreciated that there remains a need in the art for a simple, effective means to overcome the difficulty of separating RNA from the protein and DNA from biological sources before the RNA is degraded by nucleases, such as RNase.

Summary of the Invention

Accordingly, in a first aspect, the present invention provides a method for isolating RNA from a biological sample, which comprises:

(i) treating the sample with a lysis solution comprising a detergent at a pH of greater than 6 to form a lysate comprising an RNA-containing liquid phase; (ii) contacting the lysate with a first solid phase bearing a positive charge in the presence of a salt so as to precipitate DNA and protein from the RNA- containing liquid phase onto the first solid phase;

(iii) separating the RNA-containing liquid phase from the solid phase;

(iv) contacting the RNA-containing liquid phase with a non-ionic solid phase in the presence of a lower alcohol comprising ethanol or propanol to precipitate RNA onto the non-ionic solid phase to form an RNA-containing solid phase;

(v) separating the RNA-containing solid phase from the liquid phase; and

(vi) isolating RNA therefrom.

It has been found that a combination of steps including a step (ii) which favours precipitation of DNA and protein, and step (iv) which favours precipitation of RNA enables a relatively simple means for isolating RNA from contaminants typically found in biological samples. As described in further detail herein, the use of solid phases in these steps allows the method to be readily automated.

The present invention is applicable to a variety of starting materials which may contain a mixture of biological substances such as proteins, carbohydrates, lipids and nucleic acids. Such biological samples include complex mixtures of these components and may comprise prokaryotic or eukaryotic cells including cultured cells, cellular inclusions such as mitochondria, viruses, protozoa, fungi, other microbes, and fresh or preserved tissue samples. Biological samples also include biological fluids, for example blood and cerebrospinal fluid.

In step (i) of the method according to the invention, the biological sample is treated with a lysis solution to form a lysate. The primary aim of this step is to release the RNA from its biological environment, where it may be bound to other components, so as to form a lysate comprising an RNA-containing liquid phase. For example, in cells, it would be necessary to disrupt the cell membrane and any lipid, protein, carbohydrate and DNA associated with the RNA. In certain viruses, it would be necessary to disrupt the viral coat protein. Whatever type of sample is being treated, the RNA is desirably released into the liquid phase, preferably in solution. Precipitation of RNA at this point in the method is preferably completely avoided. Accordingly, a detergent is used to treat the sample at a pH of at least 6, preferably at least 6.5, more preferably at least pH 7.0 and preferably no more than pH 9.5, more preferably no more than pH 8.5. At pHs much above pH 9 RNA degradation becomes significant and this is undesirable. At pHs below 6, the relative yield of RNA diminishes.

The detergent used in the lysis solution may be ionic or non-ionic and is preferably an anionic detergent. A particularly preferred detergent is a dodecyl sulphate, such as sodium dodecyl sulphate (SDS). The lysis solution may be buffered and may include components such as metal ion chelators such as ethylenedi amine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). However, no components should be included which are likely to precipitate RNA. High lower alcohol contents such as 70% ethanol or 50% isopropanol would precipitate most or all of the RNA. Accordingly, if alcohols are to be included in the lysis solution, they must be at a concentration insufficient to precipitate RNA. For example, 5-10% lower alcohols would be tolerated, hi a preferred embodiment, the lysis solution is substantially free of alcohols. It is further preferred that the lysis solution is substantially free of chaotropic salts.

In step (ii), the lysate is contacted with a first solid phase bearing a positive charge in the presence of a salt so as to precipitate DNA and protein. Conditions may be selected so as to precipitate DNA and protein selectively without substantial precipitation of RNA. This enables subsequent removal of the solid phase without substantial removal of the RNA target material.

A positively charged solid phase is used and such solid phases include positively charged or electropositive surfaces such as positively functionalised silica, positively functionalised silanol, positively functionalised metal oxides, positively functionalised vinyl polymers, such as polyvinyl alcohol, and other positively charged polymeric surfaces. An aminated surface is particularly preferred because it binds well to DNA without substantial RNA removal. Preferably, the first solid phase comprises polyvinyl alcohol functionalised with amine groups. In another preferred embodiment the first solid phase comprises iron oxide functionalised with amine groups. The positively functionalised solid phase may comprise chitosan, a naturally occurring polysaccharide comprising amine groups, or other naturally occurring polymers which bear positively-charged groups. The solid phase is positively charged at the pH at which the lysate is contacted.

The lysate and first solid phase are contacted in the presence of a salt so as to precipitate DNA and protein from the RNA-containing liquid phase. Both organic and inorganic salts may be used in this step, preferably monovalent salts. Sodium salts, potassium salts and ammonium salts have been found to be particularly useful, especially ammonium salts such as ammonium acetate. The concentration of salt must be sufficient to precipitate the DNA and protein and a concentration of at least 1 molar is preferred. It may be necessary to contact the lysate with the first solid phase for a period from 10 seconds to 20 minutes to achieve precipitation. Typically proteins and genomic or high molecular weight DNA will precipitate in this step onto the solid surface of the first solid phase. Without wishing to be bound by theory, the inventors understand that the DNA and protein bind to the solid phase in the form of a protein-DNA complex, such as for example chromatin.

The form which the first solid phase takes will depend upon the apparatus to be used to perform the method of the invention. The solid phase may take the form of a filter, column, non-magnetic particles, or magnetic particles such as a ferrimagnetic or paramagnetic particles. Magnetic particles may be used in a magnetic separation apparatus, which may be an automated magnetic separation apparatus, as discussed in further detail below.

According to step (iii), the RNA-containing liquid phase is separated from the solid phase. Such separation may be effected by any conventional means. For example, separation may be effected by centrifugation or pressure/vacuum or gravity in the case of columns; pressure/vacuum in the case of filters; centrifugation or gravity in the case of non-magnetic particles; and magnetic separation in the case of magnetic particles.

In step (iv), the RNA-containing liquid phase is contacted with a non-ionic solid phase in the presence of a lower alcohol to precipitate RNA onto the non-ionic solid phase to form an RNA-containing solid phase. It is preferred that conditions are selected so as to precipitate RNA selectively without substantial precipitation of any residual DNA in the RNA-containing liquid phase. This enables subsequent isolation of the RNA-containing solid phase with contaminants remaining in the liquid phase. A non-ionic solid phase is used in this step because a positively charged surface binds little RNA or DNA under these conditions and a negatively-charged surface binds both RNA and DNA. By selecting a suitable non-ionic surface it has been found that the nucleic acid that binds thereto in the presence of a lower alcohol comprising ethanol or propanol consists of RNA; RNA binds preferably to DNA. The non-ionic solid phase preferably comprises a non-ionic surface, which is preferably a surface with polar or non-polar non-ionic functional groups. Particularly preferred surfaces are those which comprise functional groups of the hydroxyl or ether type. A preferred surface comprising ether groups is an epoxy surface.

The step of contacting the RNA-containing liquid phase with a non-ionic solid phase takes place in the presence of a lower alcohol comprising ethanol or propanol. The concentration of the lower alcohol is preferably sufficient to precipitate the RNA onto the non-ionic solid phase without substantial precipitation of DNA. A preferred amount of ethanol used in this step is in the range of from 1 to 2.5 vol/vol RNA- containing liquid phase. If propanol is used in this step, this is preferably isopropanol, although n-propanol could be used. A preferred amount of isopropanol is in the range of 0.5 to 1 vol/vol RNA-containing liquid phase.

As with the first solid phase, the non- ionic solid phase may be in the form of a filter, column, non-magnetic particle or magnetic particle such as a ferrimagnetic or paramagnetic particle. Again, the choice of solid phase will depend upon the apparatus used to perform the method.

In step (v), the RNA-containing solid phase is separated from the liquid phase. In common with step (iii), step (v) is also conventional and will depend upon the form of the non-ionic solid phase. For example, separation may be effected by centrifugation or pressure/vacuum or gravity in the case of columns; pressure/vacuum in the case of filters; centrifugation or gravity in the case of non-magnetic particles; and magnetic separation in the case of magnetic particles. Once the RNA-containing solid phase is separated from the liquid phase the RNA is isolated therefrom according to step (vi) of the invention. Conventional methods may be employed in this step. The step may comprise one or more steps of washing the RNA-containing solid phase and a step of eluting RNA from the RNA-containing solid phase. In one embodiment, a first washing solution comprising 70% ethanol or 50% isopropanol is applied to the RNA-containing solid phase from step (v) in a first washing step. In a second washing step, a second washing buffer is applied, which comprises a mixture of a chaotropic salt and a lower alcohol such as ethanol or isopropanol, typically at a concentration of 20 to 40%.

The RNA may be eluted from the solid phase into an elution solution containing low salt concentrations. In a preferred embodiment, 0.1 to 10 mM Tris-HCl may be used at a slightly alkaline pH e.g. pH 8.0 or, alternatively, water.

In a particularly preferred embodiment, the first solid phase and the second, non-ionic solid phase both comprise magnetic particles and at least steps (ii) to (vi) are performed on an automated magnetic separation apparatus. The automated magnetic separation apparatus may include a computer which processes data comprising instructions for operating steps of the method, such as at least steps (ii) to (vi). One type of magnetic separation apparatus is disclosed, for example, in GB 0717452.7, filed on 7¹ September 2007 by the present applicants. This and similar apparatus typically includes an automated pipette head assembly movable within the apparatus so that it may be aligned with test tubes or vials for reagent liquid handling. A step of magnetic separation may be performed using a magnetic rod which is also movable within the apparatus for alignment with test tubes or vials. Alignment steps and steps of liquid aspiration and liquid dispensing may all be controlled by a computer such as a micro computer which is linked to or forms part of the automated magnetic separation apparatus. Data comprising instructions for operating steps of the present method may be processed by the computer. The data may be provided to the computer from a removable data carrier such as a compact disc or through a communications network from a remote computer. For example, the remote computer may be a server which may be linked to the computer through the internet thereby allowing data comprising the instructions to be downloaded for use by the apparatus. In a further aspect, the present invention provides a remote computer or a removable data carrier, configured to provide data comprising instructions for operating steps of the method as described herein.

In a further aspect, the present invention provides a kit for use in the method as described herein. The kit comprises the following components:

(a) a lysis solution comprising a detergent at a pH of greater than 6 for forming from a biological sample a lysate comprising an RNA-containing liquid phase;

(b) a first solid phase bearing a positive charge;

(c) a salt solution for precipitating DNA and protein from the RNA- containing liquid phase in the presence of the first solid phase;

(d) a non-ionic solid phase;

(e) a lower alcohol comprising ethanol or propanol for precipitating RNA onto the non-ionic solid phase to form an RNA-containing solid phase;

(f) optionally one or more washing solutions for washing the RNA- containing solid phase; and

(g) optionally an elution solution to elute RNA from the RNA-containing solid phase.

The exact form of the kit will depend upon how the method is to be operated. Where the method is to be operated manually, for example using as solid phases filters, columns or non-magnetic particles, each component of the kit may be supplied in a separate container. Where the method is automated, components of the kit may be supplied in a manner to be used with appropriate apparatus. For example, in a preferred magnetic separation apparatus the kit is supplied with solid phases in the form of magnetic particles. With the optional exception of the lysis solution, the components may be supplied in a plurality of containers forming a cartridge for use in the apparatus. The lysis solution may be supplied in a separate container, if required. Typically such cartridges are made of disposable plastics and the user of the apparatus uses one cartridge for each biological sample. Where the non-ionic solid phase comprises magnetic particles, they may be supplied together with the lower alcohol in the same container. Similarly, the first solid phase and salt solution may be supplied together in the same container where the first solid phase comprises magnetic particles. In an automated magnetic separation apparatus, combining a solid phase with its associated solution allows the solid phase and solution to be aspirated together from an individual container.

Detailed Description of the Invention

The present invention will now be described in further detail, by way of example only, with reference to the accompanying drawings, in which:

FIGURE 1 shows the results of agarose gel electrophoresis on samples to compare solid phase ability to precipitate DNA;

FIGURE 2 shows the results of agarose gel electrophoresis on samples to compare solid phase ability to precipitate RNA; and

FIGURE 3 shows the results of agarose gel electrophoresis on samples to compare aminated solid phase ability to precipitate DNA.

Examples

Example 1

This example demonstrates that the salt precipitation step (ii) of the invention, following the lysis step (i), exhibits specificity that depends on the surface of the first solid phase.

Experimental procedure

1. Disrupt 1 mill HeLa cells in 300μl 0,5%SDS/50mM EDTA, pH 8,5

2. Mix 10 cycles, leave alone for 1 min

3. Add lOOμl 5M ammonium acetate, pH 7 with 5mg test beads (carboxylated polyvinylalcohol (PVA) surface or aminated PVA surface) - salt precipitation step (ii)

4. Mix 10 cycles, leave alone for 5 min

5. Collect beads using magnet. Transfer supernatant to fresh tube and discard beads.

6. Add 350 μl isopropanol containing 2,5mg unfunctionalized PVA beads, leave alone for 5 min - RNA precipitation step (iv)

7. Collect beads using magnet. Aspirate and discard supernatant 8. Wash beads 5 times (resuspend beads and recollect them on magnet)

9. Resuspend beads in 200 μl RNase free water, leave to stand for 5 min

10. Collect beads using magnet. Transfer supernatant to fresh tube and discard beads.

11. Mix 5μl of all the samples + 5μl 2X Loading dye, place at 70⁰C for 10 min, cool on ice. Load 9μl of mix to a 1% agarose gel w/EtBr

12. Run gel at 80V for 1,5 hour

Figure 1 shows a gel following the experimental procedure. The preparations that have been subjected to electrophoresis have been through all of the steps of the complete isolation method. They have therefore been through the non-ionic precipitation step (iv) that shows preference for RNA as well. All samples have been treated in the same way except for the different beads in the salt precipitation. The results demonstrate that some solid phases can selectively remove more DNA than others at the salt precipitation step. The negatively charged carboxylated beads do not remove as much DNA at the salt precipitation step (ii) as the positively-charged aminated beads do.

Example 2

This example demonstrates that the RNA precipitation step (iv) has a specificity that depends on the surface of the solid phase.

Experimental procedure: The samples were processed following the method described in Example 1 , with the exception that aminated PVA beads only were used during the salt precipitation step and either aminated, plain (unfunctionalized) or carboxylated PVA beads were used during the RNA precipitation step.

Figure 2 shows a gel following the experimental procedure. The preparations that have been subjected to electrophoresis have been through all of the steps of the complete isolation method. They have therefore been through the salt precipitation step (ii) that removes some of the DNA. All samples have been treated in the same way except for the different beads in the RNA precipitation step (iv). Lanes 3, 4 and 5 correspond respectively to RNA preparations obtained in the above procedure in which the RNA precipitation step used: carboxylated beads (3), unfunctionalised PVA beads (4) and aminated beads (5). The results demonstrate that solid phases can exhibit specificity for RNA: the unfunctionalized beads yield RNA without contaminating DNA being detectable on the gel.

Example 3

This example relates to a preferred embodiment of the invention.

Experimental procedure

1. Disrupt 1 mill HeLa cells in 300μl 0,5%SDS, pH 8,5

2. Mix 10 cycles, leave alone for 3 min

3. Add lOOμl 5M ammonium acetate with 2,5mg aminated PVA beads

4. Mix 15 cycles, leave alone for 5 min

6. Add 400 μl isopropanol containing 2,5mg epoxy functionalized PVA beads, leave alone for 5 min

7. Collect beads using magnet. Aspirate and discard supernatant

8. Wash beads 5 times (resuspend beads and recollect them on magnet)

9. Resuspend beads in 100 μl RNase free water, leave alone for 2,5 min

13. Mix 5μl of all the samples + 5μl 2X Loading dye, place at 70°C for 10 min, cool on ice. Load 9μl of mix to a 1% agarose gel w/EtBr

14. Run gel at 80V for 1,5 hour

15. Optical Density (OD) at 260 nm and 280nm wavelength was measured using a Perkin Elmer Lambda spectrophotometer. A ratio of approximately 2.1 between OD at 260 nm and at 280 nm is an indication of pure RNA

16. Concentrations were calculated from Optical Density values according to the formula (OD at 260 nm) X 40 μg/ml

17. Ct values were obtained using an Applied Bio systems 7300 with Hs_ACTB_SG_l QuantiTect Primer Assay and Quantitect SYBR Green RT- PCR Kit, one step, both kits from QIAGEN NV according to the manufacturer's instructions. Ct is the number of PCR cycles necessary to produce a threshold level of product. Delta Ct is the difference in Ct between a reaction based on RNA and DNA in the isolate. The data in Table 1 indicate that there is a 16 000 fold excess of RNA templates compared to DNA templates in the isolations.

18. 5 μl of the isolates were loaded on to RNA columns on an Agilent BioAnalyser. The BioAnalyser gives an estimate of the concentration, a ratio of the band intensity between the 28S and 18S ribosomal RNA bands (between 1,6 and 2,2 is considered good) and an RNA Integrity Number based on a broader measurement (0 to 10, where values above 8 is considered good). The results are shown in Table 2.

Example 4

This example demonstrates that surfaces with related physical properties have the same specificity in step ii of the invention.

Experimental procedure

Disrupt 1 mill HeLa cells in 300μl 0,5%SDS/50mM EDTA, pH 8,5

Mix 15 cycles, leave alone for 1 min

Add lOOμl 5M ammonium acetate with 5mg test beads

Test bead 1 : amine functionalized PVA

Test bead 2: amine functionalized iron oxide

Test bead 3: chitosan beads

Test bead 4: negative control (bead that does not bind DNA) Mix 10 cycles, leave alone for 5 min

Collect beads using magnet. Transfer supernatant to fresh tube and discard beads. Add 800 μl ethanol containing 2,5mg epoxy functionalized PVA beads, leave alone for 5 min

Collect beads using magnet. Aspirate and discard supernatant Wash beads 5 times (resuspend beads and recollect them on magnet) Resuspend beads in 200 μl RNase free water, leave alone for 2,5 min Collect beads using magnet. Transfer supernatant to fresh tube and discard beads. Mix 5μl of all the samples + 5μl 2X Loading dye, place at 70⁰C for 10 min, cool on ice. Load 9μl of mix to a 1% agarose gel w/EtBr Run gel at 80V for 1,5 hour

Figure 3 shows the results of the agarose gel electrophoresis. Table 1 Average data from 8 samples

Table 2: Analysis results from the Agilent Bio Analyser

Claims

1. A method for isolating RNA from a biological sample, which comprises:

(i) treating the sample with a lysis solution comprising a detergent at a pH of greater than 6 to form a lysate comprising an RNA-containing liquid phase;

(ii) contacting the lysate with a first solid phase bearing a positive charge in the presence of a salt so as to precipitate DNA and protein from the RNA- containing liquid phase onto the first solid phase;

(iii) separating the RNA-containing liquid phase from the solid phase;

(iv) contacting the RNA-containing liquid, phase with a non-ionic solid phase in the presence of a lower alcohol comprising ethanol or propanol to precipitate RNA onto the non-ionic solid phase to form an RNA-containing solid phase;

(v) separating the RNA-containing solid phase from the liquid phase; and

(vi) isolating RNA therefrom.

2. A method according to claim 1, wherein the sample is treated with the lysis solution at a pH in the range of from 6.5 to 9.5.

3. A method according to claim 1 or claim 2, wherein the detergent comprises an anionic detergent.

4. A method according to claim 3, wherein the anionic detergent is a dodecyl sulphate.

5. A method according to any preceding claim, wherein the lysis solution is substantially free of alcohols.

6. A method according to any of the claims 1 through 4, wherein the lysis solution is substantially free of chaotropic salts.

7. A method according to any preceding claim, wherein the first solid phase is a positively functionalised solid phase.

8. A method according to claim 7, wherein the first solid phase comprises an aminated surface.

9. A method according to any preceding claim, wherein the first solid phase comprises chitosan.

10. A method according to claim 7 or claim 8, wherein the first solid phase comprises a positively functionalised vinyl polymer or a positively fbnctionalised metal oxide.

11. A method according to any preceding claim, wherein the salt in step (ii) is a monovalent salt.

12. A method according to claim 11, wherein the monovalent salt is an ammonium salt.

13. A method according to any preceding claim, wherein the salt in step (ii) is present at a concentration of at least IM.

14. A method according to any preceding claim, wherein the non-ionic solid phase comprises a non-ionic surface comprising hydroxy or ether groups.

15. A method according to claim 14 wherein the non-ionic solid phase comprises an epoxy surface,

16. A method according to any preceding claim, wherein the lower alcohol of step (iv) comprises: (a) ethanol in an amount in the range of from 1 to 2.5 vol/vol RNA- containing liquid phase; or (b) isopropanol in an amount in the range of from 0.5 to 1 vol/vol RNA- containing liquid phase.

17. A method according to any preceding claim, wherein the step (vi) of isolating RNA comprises one or more steps of washing the RNA-containing solid phase; and a step of eluting RNA from the RNA-containing solid phase.

18. A method according to any preceding claim, wherein the first solid phase and/or the non-ionic solid phase comprises magnetic particles.

19. A method according to claim 18 wherein each of the first solid phase and the non-ionic solid phase comprises magnetic particles and at least steps (ii) to (vi) are performed on an automated magnetic separation apparatus.

20. A method according to claim 19, wherein the automated magnetic separation apparatus includes a computer which processes data comprising instructions for operating steps of the method.

21. A method according to claim 20, wherein the data are provided to the computer through a communications network from a remote computer or from a removable data carrier.

22. A remote computer or a removable data carrier, configured to provide data comprising instructions for operating steps of the method according to claim 20.

23. A kit for use in a method for isolating RNA according to any of claims 1 to 21, which comprises the components:

(b) a first solid phase bearing a positive charge;

(d) a non-ionic solid phase;

24. A kit according to claim 23, wherein the lysis solution has a pH in the range of

25. A kit according to claim 23 or claim 24, wherein the detergent comprises an anionic detergent.

26. A kit according to claim 25, wherein the anionic detergent is a dodecyl sulphate.

27. A kit according to any one of claims 23 to 26, wherein the lysis solution is substantially free of alcohols.

28. A kit according to any one of claims 23 to 26, wherein the lysis solution is substantially free of chaotropic salts.

29. A kit according to any one of claims 23 to 28, wherein the first solid phase is a positively functionalised solid phase.

30. A kit according to claim 29, wherein the first solid phase comprises an animated surface.

31. A kit according to any of claims 23 to 30, wherein the first solid phase comprises chitosan.

32. A kit according to claim 29 or claim 30, wherein the first solid phase comprises a positively functionalised vinyl polymer or a positively functionalised metal oxide.

33. A kit according to any one of claims 23 to 32, wherein the salt in step (ii) is a monovalent salt.

34. A kit according to claim 33, wherein the monovalent salt is an ammonium salt.

35. A kit according to any one of claims 23 to 34, wherein the salt is present at a concentration of at least IM.

36. A kit according to any one of claims 23 to 35, wherein the non-ionic solid phase comprises a non-ionic surface comprising hydroxy or ether groups.

37. A kit according to claim 36 wherein the non-ionic solid phase comprises an epoxy surface.

38. A kit according to any one of claims 23 to 37, wherein the lower alcohol comprises: ethanol in an amount in the range of from 1 to 2.5 vol/vol RNA-containing liquid phase; or isopropanol in an amount in the range of from 0.5 to 1 vol/vol RNA- containing liquid phase.

39. A kit according to any one of claims 23 to 38, wherein the first solid phase and/or the non-ionic solid phase comprises magnetic particles.

40. A kit according to claim 39, wherein the non-ionic solid phase and lower alcohol are supplied together in the same container.

41. A kit according to claim 39 or claim 40, wherein the first solid phase and salt solution are supplied together in the same container.

42. A kit according to any one of claims 39 to 41, wherein components (b) to (f) of the kit are provided in a cartridge comprising a plurality of containers, for use in an automated magnetic separation apparatus.