WO2017203225A1

WO2017203225A1 - Isolating nucleic acids

Info

Publication number: WO2017203225A1
Application number: PCT/GB2017/051428
Authority: WO
Inventors: Stephen John Minter; Georgios Patsos
Original assignee: RevoluGen Limited
Priority date: 2016-05-24
Filing date: 2017-05-22
Publication date: 2017-11-30
Also published as: GB201609115D0

Abstract

A method of extracting RNA substantially free from DNA from biological material susceptible to lysis, the method comprises the steps of: (a) effecting lysis of said biological material with a lysis solution which comprises water, dissolved lithium at a concentration of 0.5M to 1.0M, and 20-60% by volume in the solution of miscible alkanol to produce a lysed composition; (b) treating the lysed composition with a support capable of immobilising RNA; (c) separating the solid support with immobilised RNA from the liquid of the lysed composition; (d) effecting washing with a first wash solution comprising water, 20-90% by volume in the first wash solution of miscible alkanol, and optionally dissolved lithium at a concentration up to 3M, and subsequently with a second wash solution which comprises at least 80% by volume of alkanol, the balance if any being water; and (e) eluting RNA from the washed solid support.

Description

Isolating Nucleic Acids

The present invention relates to the isolation of RNA from biological material (e.g. cells) by use of a lysis procedure and processing of the lysate to isolate RNA. More particularly, the invention involves the immobilisation of nucleic acids comprising RNA from the lysed biological material on a solid support and purification of the RNA on the support prior to elution of the RNA from the support for collection. A particular aim of the present invention is to provide a process which is relatively quick and straightforward to practise and which produces RNA of high purity in good yield. The invention also relates to compositions for use in the lysis procedure.

The isolation of pure, intact RNA from biological material (e.g. cells) is of importance in a number of fields, e.g. research and clinical diagnosis. Thus, purely by way of example, clinical cell-containing samples from a patient may be subjected to a procedure (including the step of cell lysis) to isolate RNA which is then analysed by procedures well known in the art. Such analysis may be for the purpose of identifying RNA from a particular bacterium (to determine whether or not the patient has been infected by that bacterium). According to a first aspect of the present invention there is provided a method of extracting RNA substantially free from DNA from biological susceptible to lysis, the method comprising the steps of:

(i) effecting lysis of said biological material with a lysis solution which comprises water, dissolved lithium at a concentration of 0.5M to 1 .0M, and 20-60% by volume in the solution of miscible alkanol to produce a lysed composition;

(ii) treating the lysed composition with a support capable of immobilising RNA; (iii) separating the solid support with immobilised RNA from the liquid of the lysed composition;

(iv) treating the solid support with immobilised RNA with a first wash solution comprising water, 20-90% by volume in the first wash solution of miscible alkanol, and optionally dissolved lithium at a concentration up to 3M; (v) separating the solid support from the first wash solution;

(vi) treating the solid support with a second wash solution which comprises at least 80% by volume of alkanol, the balance if any being water;

(vii) separating the solid support from the second wash solution; and

(viii) eluting RNA from the solid support.

Throughout this specification, the term "comprising" or "comprise(s)" means including the component(s) specified but not to the exclusion of the presence of other components. The term "consisting essentially of" or "consists essentially of" means including the components specified but excluding other components except for materials present as impurities and unavoidable materials present as a result of processes used to provide the components. Typically, a composition "consisting essentially of" a set of components may comprise less than 5% by weight, more typically less than 1 % by weight of non-specified components. The invention provides excellent results in the isolation of RNA. The isolated RNA is highly pure, there being little risk of contamination by lithium salts in view of the relatively low amounts thereof employed in the process of the invention. The method of the invention results in the isolation of substantially pure and substantially intact RNA.

The method of the invention may be used for isolating RNA from a wide range of RNA- containing samples of biological origin. The sample from which the RNA is to be isolated may be, or may comprise, cells, for example animal (including mammalian) cells, plant cells, bacterial cells, or yeast or other fungal cells. Alternatively, the sample from which the RNA is to be isolated may be, or may comprise, one or more viruses. The cells, virus(es) or other RNA-containing sample from which the RNA is to be isolated may originate from or have been present in a tissue sample or body fluids such as blood, sputum, urine or CSF In certain embodiments (e.g. for the lysis of gram negative or gram positive bacteria), the sample may additionally be treated with lysozyme to "weaken" the cell walls prior to lysis. In step (i) of the method of the invention for isolating RNA substantially free from DNA from cells, the biological material (e.g. cells) is lysed with a lysis solution which comprises water, dissolved lithium (present as ions) at a concentration of 0.5M to 1 .OM and 20-60% by volume in the solution of miscible alkanol, so as to release RNA from the material. It is preferred that the lysis solution is "pre-formulated" so that the lysis solution of the desired composition may be added to the biological material. However, we do not preclude the possibility that the components of the lysis solution are added individually to the biological material in the requisite amounts. The lysis solution may comprise the stated components, although should be free of agents that prevent nucleases breaking down DNA. It is particularly preferred that the lysis solution does not include any anionic surfactant (so the lysis solution preferably does not include any SDS). The lysis solution may consist essentially of the stated components, or consist of these components. Most preferably, the lysis solution consists essentially of water, at least one lithium salt such that the total lithium concentration is 0.5M to 1 .0M, and 20-60% by volume of miscible C^₃ alkanol. The lithium salt preferably comprises a lithium halide, which is preferably the sole lithium salt in the lysis solution. The lithium salt is preferably lithium chloride.

Preferably, the concentration of lithium in the lysis solution (e.g. provided by lithium chloride as the sole lithium salt) is 0.7M to 1 .0M, and even more preferably 0.85M to 0.95M. A concentration of about 0.9M is particularly preferred. The lysis solution preferably comprises 35% to 45% by volume of miscible alkanol, which is preferably provided by ethanol and/or isopropanol (which if present together make up the stated concentration range). More preferably, the lysis solution comprises about 40% by volume of miscible alkanol. It is particularly preferred that the miscible alkanol is iso-propanol (present as the sole miscible alkanol). The lysis procedure will generally involve thoroughly admixing the biological material to be lysed with the lysis solution (e.g. by pipetting). Lysis may be effected at ambient temperature (e.g. 15°C to 25°C) without the need for additional heating. A time period of 15-25 minutes, e.g. about 20 minutes, is generally suitable to produce a lysed composition. The lysed composition is treated with a solid support capable of immobilising RNA. The solid support is preferably a porous material. The solid support as used for the method of the invention may be a silica or silica-based material. The silica or silica- based material may be a glass, preferably a borosilicate glass. The support may comprise a fibrous material, and preferably comprises fibres of a silica or silica-based material. The filter preferably comprises borosilicate glass fibres. For preference, the support is in the form of a fibrous sheet or membrane in which the fibres are of a borosilicate glass. Filters of the type available under the designation POREX F are suitable for use in the invention. Conveniently, the solid support is provided as a filter (in the form of a sheet or membrane) in a spin tube of the type well known in the art. As indicated, the filter in the spin tube is preferably a sheet or membrane comprising borosilicate fibres. Preferably more than one such filter or sheet is provided in the spin tube. Most preferably, there are four such membrane or sheet filters (in face-to-face relationship) in the spin tube. Particularly good results are obtained where the sheet or membrane filters comprise the material available as POREX F.

The lysed composition resulting from step (i) of the method may be introduced into the spin tube which is then centrifuged (although we do not preclude the possibility that the lysis treatment of step (i) may be effected in the spin tube itself). During centrifugation, the lysed composition passes through the porous support and is thereby treated whereby the nucleic acids comprising RNA (that is ultimately to be isolated) become bound to the solid support and the majority of impurities (proteins etc.) pass through the solid support (filter) and are collected in the supernatant, which may then be discarded.

Without wishing to be bound by theory, we believe that the absence, from the lysis solution, of agents (e.g. SDS) that prevent nucleases breaking down DNA ensures that DNA is degraded and is not captured on the support, whereby only RNA is immobilised.

In the next step of the method, the solid support is washed to remove any remaining impurities and leave substantially pure RNA immobilised on the support. Washing is effected in two stages. In the first wash stage (step (iv) of the method), the solid support is treated with a first wash solution containing water, 20-90% (preferably 20- 80%) by volume in the first wash solution of miscible alkanol, and optionally dissolved lithium in a concentration up to 3M.

The first wash solution may comprise the stated components, may consist essentially of the stated components, or consist of these components. In one embodiment of the invention, the wash solution may consist essentially of (or consist of) water and 60- 80%, preferably 65-75%, most preferably about 70% by volume of miscible alkanol. In a further embodiment of the invention, the first wash solution consists essentially of water, at least one lithium salt such that the total lithium concentration is up to 3M, and 20-60% by volume of miscible alkanol. The lithium salt (if used) preferably comprises a lithium halide which is preferably the sole lithium salt in the first wash solution. The lithium salt is preferably lithium chloride.

Preferably, the concentration of lithium (if used) in the first wash solution (e.g. provided by lithium chloride as the sole lithium salt) is 0.1 M to 2.5M. A concentration of about 0.4M-0.6Mm, most preferably about 0.5M, is particularly preferred. The first wash solution which contains lithium preferably comprises 45% to 55% by volume of miscible C!-3 alkanol, preferably about 50% by volume. It is particularly preferred that the miscible alkanol in the first wash solution comprises ethanol, most preferably as the sole miscible C 3 alkanol in the first wash solution.

If the isolation procedure is being carried out in a spin tube, then the first wash solution is added to the spin tube which is then centrifuged to cause the first wash solution to pass through the filter and remove at least some of the remaining impurities for collection in the supernatant, which is then discarded.

A second, and usually final, wash step is then effected with a second wash solution which comprises at least 80% by volume of alkanol, the balance if any being water. The second wash solution preferably consists essentially of alkanol and water. The concentration of alkanol in the second wash solution is preferably 85%-95% by volume, an amount of about 90% by volume being particularly preferred. The alkanol in the second wash solution preferably comprises ethanol and/or isopropanol, and is most preferably ethanol present as the sole alkanol. In the case where the method is effected using a spin tube, the second wash liquid is added to the spin tube which is then centrifuged to force the wash liquid through the filter and remove remaining impurities for collection in the supernatant, which is then discarded.

At this stage, the desired RNA is immobilised, in substantially pure form, on the solid support and can be eluted with an elution solution. The elution solution may comprise (consists essentially of or consists of) a buffered solution of EDTA or a salt thereof. A particular suitable elution solution comprises Tris-HCI in a concentration of about 10mM, and EDTA disodium salt dihydrate at a concentration of about 0.5mM, the solution having a pH of 9. As an alternative, RNase-free water can be used for elution.

In the case where the method of the invention is being practised with a spin-column containing a filter on which the RNA is immobilised, the elution solution may be added to the spin column which is then centrifuged to cause the elution solution to elute the nucleic acids comprising RNA from the solid support for collection.

The collected nucleic acids comprising RNA may then be further analysed as required.

The RNA obtained by the method of the invention is sufficiently pure to be used for the purpose of further analysis by techniques well known in the art. The RNA may be used, for example, for the purposes of research or for diagnosis of a medical condition, as required.

The method of the invention may conveniently be effected on a pellet of the biological material (e.g. cells) to be lysed. The pellet may be produced by centrifugation of a liquid sample containing the biological material, using techniques well known in the art. This invention will be illustrated by the following non-limiting Example and the accompanying drawings, in which:

Fig. 1 shows the results of gel electrophoresis on the product obtained in Example 1 below; and Fig. 2 shows the results of Example 2. Example 1

This Example demonstrates extraction of RNA from a suspension containing ca 10⁶ HeLa cells.

The suspension was centrifuged to yield a pellet which was then re-suspended in 600μΙ of a lysis solution (0.9M lithium chloride in 40% iso-propanol). The admixture was then incubated at room temperature for 20 minutes before the full volume was transferred to a spin column having 4 POREX F filters. The column was spun at 8000 rpm for 1 minute (although any spin-speed of 8000 to 1 1 ,000 rpm would be suitable). The "flow- through" from the column was then discarded.

To the column was then added a first wash solution (2M LiCI in 50% ethanol) and the column was centrifuged at 8000 rpm for 1 minute. "Flow-through from the column was discarded.

500μΙ of a second wash solution (90% ethanol) was then added to the spin-column which was then centrifuged at 14,000 rpm for 3 minutes prior to the "flow-through" being discarded.

A further spin at 14,000 rpm for 1 minute was then performed to remove all residual alcohol. 150μΙ of elution buffer (10mM Tris-HCI, 0.5mM EDTA, pH 9.0) were then added to the column (a volume in range of 100μΙ-200μΙ would have been equally suitable) prior to the column being incubated at room temperature for 1 minute and then spun at 8000 rpm for 1 minute. The "flow-through", which is an RNA extract obtained from the HeLa cells was collected in 1 .5ml Eppendorf tubes.

5μΙ aliquots of the RNA extract were loaded onto a 1 % ethidium bromide agarose gel tog3ether with a Hind III ladder. The result is shown in Fig. 1 (in which bp = base pairs). Fig. 1 clearly demonstrates intense bands with minimal degradation. All replicates exhibited the typical RNA phenotype. No DNA was present but two ribosomal RNA bands can clearly been seen.

2μΙ aliquots of the RNA extract were quantified by nanodrop spectrophotometry. The results are shown in Table 1 below:

Table 1

In general, an A260/A280 value of 2 to 2.2 is considered pure for RNA.

This Example demonstrates that the invention provides a simple and quick technique that extracts RNA without DNA contamination evident on a gel.

Example 2

5 μΙ of NEB microRNA ladder (microRNA Marker #N2102), were spiked in 600 μΙ of lysis solution (0.9M LiCI, 40% isopropanol) in four replicates. Each admixture was then incubated at room temperature for 20 minutes before the full volumes were transferred to individual spin columns each having 4 POREX F filters. The columns were spun at 8000 rpm for 1 minute (although any spin-speed of 8000 to 1 1 ,000 rpm would be suitable). The "flow-through" from the column was then discarded. Two of the replicates were washed with 70% ethanol (not containing lithium chloride) as a first wash solution whereas the other two replicates were washed with 0.4M LiCI in 50% ethanol as their first wash solution.

500μΙ of a second wash solution (90% ethanol) were then added to the individual spin- columns which were then centrifuged at 14,000 rpm for 3 minutes prior to the "flow- through" being discarded. An extra spin at 14,000 rpm for 1 minute was performed. This is a dry spin to get rid of all residual alcohol.

All replicates were eluted in 200 μΙ of RNase free H₂0, and were lyophilised. The lyophilised materials were each solubilised in 10 μΙ of elution buffer (10mM Tris-HCI, 0.5mM EDTA, pH9.0). (The reason for lyophilisation was to concentrate the material so when running the output on a gel it would be possible to compare to an input control. Since the elution was effected with 200 μΙ elution buffer and only up to 15 μΙ can be ran on a gel the material had be concentrated. In this way it was possible to visualize how much of a 5 μΙ input could be isolated in total.)

The solubilised eluates together with 5 μΙ of the input synthetic microRNAs (made up to 10 μΙ in the elution buffer) were run on a 15% Urea-PAGE gel. The image generated is shown in Fig. 2 and reveals that for all three synthetic microRNAs recovery yield was higher when the 70% Ethanol, 0M LiCI wash solution was used, instead of 0.4M LiCI in 50% ethanol.

Example 3

A modification of the procedure described in Example 1 can be used to obtain RNA from gram positive or gram negative bacteria. In this modified Example, the bacterial cells are pelleted, then treated in 100 μΙ of 3mg/ml lysozyme solution (20mM Tris, 2mM EDTA, 1 .2% Triton x-100), and then incubated at ambient temperature for 10 mins prior to addition of the lysis solution. The subsequent steps are as described in Example 1.

Claims

1 . A method of extracting RNA substantially free from DNA from biological material susceptible to lysis, the method comprising the steps of:

(ii) treating the lysed composition with a support capable of immobilising RNA;

(iii) separating the solid support with immobilised RNA from the liquid of the lysed composition;

(iv) treating the solid support with immobilised RNA with a first wash solution comprising water, 20-90% by volume in the first wash solution of miscible alkanol, and optionally dissolved lithium at a concentration up to 3M;

(v) separating the solid support from the first wash solution;

(vii) separating the solid support from the second wash solution; and

(viii) eluting RNA from the solid support.

2. A method as claimed in claim 1 wherein the lysis solution consists essentially of water, at least one lithium salt such that the total lithium concentration is 0.7M to 1 .0M, and 20-60% by volume of the miscible alkanol.

3. A method as claimed in claim 2 wherein the lithium salt comprises lithium chloride, preferably as the sole lithium salt.

4. A method as claimed in any one of claims 1 to 3 wherein the concentration of lithium in the lysis solution is in the range 0.7M to 1 .0M.

5. A method as claimed in claim 4 wherein the concentration of lithium in the lysis solution is in the range 0.85M to 0.95M.

6. A method as claimed in any one of claims 1 to 3 wherein the lysis solution comprises 35% to 45% by volume of miscible Ci-₃ alkanol.

7. A method as claimed in any one of claims 1 to 6 wherein the miscible C^₃ alkanol in the lysis solution comprises iso-propanol.

8. A method as claimed in claim 7 wherein iso-propanol is the sole C†.₃ alkanol in the lysis solution.

9. A method as claimed in any one of claims 1 to 8 wherein the first wash liquid consists essentially of water and 60%-80% by volume of the first wash liquid of miscible Ci-₃ alkanol.

10. A method as claimed in claim 9 wherein the first wash liquid consists essentially of water and 65%-75% by volume of the first wash liquid of miscible alkanol.

1 1 . A method as claimed in claim 10 wherein the first wash liquid consists essentially of water and about 70% by volume of the first wash liquid of miscible alkanol.

12. A method as claimed in any one of claims 1 to 8 wherein the first wash liquid consists essentially of water, at least one lithium salt such that the total lithium concentration is 0.1 M to 2.5M, and 30-70% by volume of miscible alkanol.

13. A method as claimed in claim 12 wherein the total lithium concentration in the first wash liquid is 0.4M to 0.6M.

14. A method as claimed in claim 13 wherein the total lithium concentration in the first wash liquid is about 0.5M.

15. A method as claimed in any one of claims 12 to 14 wherein the concentration by volume of miscible alkanol in the first wash liquid is 45%-55%.

16. A method as claimed in claim 15 wherein the concentration by volume of miscible alkanol in the first wash liquid is about 50%.

17. A method as claimed in any one of claims 12 to 16 wherein the lithium salt in the first wash liquid comprises lithium chloride, preferably as the sole lithium salt.

18. A method as claimed in any one of claims 1 to 17 wherein the miscible C†.₃ alkanol in the first wash liquid is ethanol.

19. A method as claimed in any one of claims 1 to 18 wherein the second wash solution consists essentially of 75% to 95% by volume of ethanol and water.

20. A method as claimed in any one of claims 1 to 19 wherein the solid support comprises silica.

21. A method as claimed in any one of claims 1 to 20 wherein step (iii) is effected in a spin-tube in which said solid support is a filter and steps (iii), (v), (vii) and (viii) are effected with centrifugation.

22. A method as claimed in claim 21 wherein the filter comprises borosilicate glass fibres.

23. A method as claimed in claim 22 wherein a plurality of said filters are provided in the spin tube.

24. A method as claimed in claim 23 wherein four filters are provided.

25. A method as claimed in any one of claims 1 to 24 which is effected at ambient temperature throughout.

26. A method as claimed in any one of claims 1 to 25 which is effected without vortexing.

27. A method as claimed in any one of claims 1 to 26 wherein the biological material from which the nucleic acids comprising DNA is to be isolated comprises cells.

28. A method as claimed in claim 27 wherein the cells are in the form of a pellet.