Patent Application
20070082354
The invention pertains to a method for isolating RNA, a support for performing the method of the invention, a method for manufacturing of the support as well as the use of the support of the invention.
The well known and established RNA isolation methods are mostly based on the usage of high concentrated solutions of chaotropic salts or organic solvents like phenol mixtures. This approach leads i.a. to an immediate inhibition of RNases. However, this approach is not applicable for a one step RNA purification. A one step approach needs the combination of an enzymatic lyses, the protection of RNA from RNases and the separation of RNA (or single stranded DNA) from dsDNA. A widely used RNase inhibitor is the ribonucleoside-vanadyl complex, which forms a transition state complex with RNases. These complexes have a very low dissociation constant (10−7 times lower than the dissociation constant of the enzyme-substrate complex) and are therefore such powerful RNase inhibitors (Berger in Methods of Enzymology, 152 (1987) 227-236, Academic Press London). In that method, 8-hydroxyquinoline was used as an indicator substance for removal of ribonucleoside-vanadyl complex. The ribosyl-vanadyl complexes have been widely used in the past, because they allow an enzymatic tissue lysis with a very rapid inactivation of RNases and a simultaneous degradation of DNA by utilizing a DNase I-treatment directly in the lysate. A disadvantage is that the vanadyl complexes are very strong inhibitors not only for RNases, but also other nucleic acid modifying enzymes like reverse transcriptases and Taq polymerase. Consequently, the inhibitors have to be carefully removed from the RNA preparation before starting any down-streaming enzymatic reaction like reverse transcription and polymerase chain reaction (PCR). A method for removal of ribosyl-vanadyl complexes used an extraction with phenol mixtures. Since the usage of phenol and other organic solvents due to their harmful properties, has been widely removed from nucleic acid purification protocols and the application of this kind of inhibitors has been omitted as well.
P. Blackburn et al. in “The Journal of Biological Chemistry”, Vol. 252. No. 15, pp. 5904-5910 (1977) disclose a soluble ribonuclease inhibitor from the human placenta which has been purified 4000-fold by a combination of ion exchange and affinity chromatography.
An object of the invention was to provide a method which allows for use of inhibitors of the RNases which can be removed under avoidance of phenol extraction.
This goal is achieved by using a method for isolating RNA from RNA containing samples wherein the RNA containing sample is treated with at least one DNase and/or another enzyme like proteases or collagenases for purification of RNA in presence of a complex of ribonucleosides and the oxovanadyl ion which is capable of inhibiting RNases present in the RNA containing samples and removing the complex of ribonucleosides and the oxovanadyl ion prior to down-stream processing of the RNA of the RNA containing sample by contacting the sample of the forgoing step with a chelating agent immobilized to a support, the chelating agent having sufficient affinity to the complex of ribonucleosides and the oxovanadyl ion so that it binds to the chemical entity and the RNA containing sample is freed from the inhibitor.
Preferably the RNA containing sample is a cell, tissue, body fluid, virus particle also in its lysed or otherwise disintegrated state.
The term “chelating agent” is well-known to the skilled person. The chelating agent is a chemical entity comprising at least one structural moiety interacting with the oxovanadyl ion and/or the ribonucleoside-oxovanadyl-complex.
The chelating agent immobilized on the support comprises for example the structural element verified in 8-hydroxyquinoline or its derivatives. Furthermore, ethylendiamintetra-acetat (EDTA), bipyridin, ethylene diamin, phenanthroline, oxalat, tartrat, dimethylglyoxime, diethylentriamin, can be used.
In another embodiment the chelating agent comprises a phosphonic acid moiety or a salt thereof. Furthermore, it may be a phosphonic acid derivative such as an amide or an ester. In still another embodiment also di-, tri-, tetra- or even higher carboxylic acids, their salts or derivatives, such as amides, esters or nitrites can be used.
According to the invention the support is a porous inorganic material selected from the group comprising inorganic metal oxides, such as oxides of aluminium, titanium, zirconium, silicon oxides, iron oxides, controlled pore glass (CPG), diatomaceous earth and combinations thereof.
Subject matter of the invention is also a support comprising an inorganic or organic polymer with immobilized chemical moieties which exhibit an affinity to inhibitors of RNases.
The support of the invention can be manufactured by contacting a reactive chemical having a moiety with affinity to a an inhibitor of RNases to the support or an activated support.
In one embodiment the surface of an inorganic support is coated with a substance obtained by polymerization of monomers having chelating functional groups which are capable to interact with the oxovanadyl and/or ribonucleoside-oxovanadyl-complex. In order to obtain spatially cross-linked polymer layers the polymerization can be performed in presence of bifunctional comonomers or oligomers. For example, the coating can be established by mixing the inorganic support with hydroxylvinylquinolin a bifunctional monomer and, if necessary, a polymerization catalyst. As monomers or bifunctional monomers or copolymers can be used organic molecules having one or two or more ethylenically unsaturated compounds or other functional groups which may be polymerized such as carboxyl groups and amides or acrylates and so on. There is a plethora of different monomers and copolymers which are suitable for coating an inorganic support and readily accessible for the skilled person. It is also possible to use an organic support which is functionalized with the respective chelating agents. If more or less inert organic materials are used methods for coating of these polymers can also be employed. The respective chemical reactions belong to the arsenal of a chemist having expertise in polymer-chemistry either organic or inorganic or both.
The invention is further illustrated by means of the following non-limiting examples.
The method is based on polymerization of vinyl monomers with chelating functional groups onto the surface of an inorganic support. In order to obtain spatially cross-linked polymer layers polymerization has been performed in presence of bifunctional divinyl compounds.
As vinyl compound 8-hydroxyvinylquinolin and as bifunctional divinyl compound divinyl benzene have been used.
To 30 g of macro-porous silica have been added under stirring 6,7 g of 8-hydroxyvinylquinolin, 0,75 g of divinyl benzene and 0,04 g of dinitrilazo-bis-isobutyric acid (as the initiator of polymerisation). Benzene has been used as solvent. The polymerisation has been performed at 70° C. for 7 h. The obtained product has been separated from the reaction mix by filtration, washed sequentially with dimethyl formamid, ethanol and a water-acetone mixture. Finally, the product has been dried in a desiccator.
The method is based on the chemical interaction of reactive functional groups inside a polymeric sorbent or inside a polymer layer on the surface of an inorganic support with monomeric organic compounds with chelating groups. In the present example macro-porous silica (30 g) with a polymer coat made from styrene-divinyl benzene has been used, whereby the copolymer represented 12% of the silica mass. The divinyl copolymer represents 7% of the mass of the copolymer. As initiator of polymerisation has been used benzoyl peroxide. The polymerisation took place by 80° C. for 7 h. This support has been treated by nitration, followed by reducing the obtained nitro-co-polymer, diazotization of received polyaminostyrene and azocoupling of the obtained product with 8-hydroxyquinolin (by mass. ratio of co-polymer and 8-HQ 1:1) by the well-known methods.
Essential Reagents:
(A) Tris I: 20 mM Tris HCl pH 7,4, 10 mM NaCl, 3 mM Magnesiumacetate
(B) Lysis buffer I: Tris I+5% (w/w) Sucrose+1,2% (w/w) Triton N-101
(C)VRC (Sigma-Aldrich 94740): 200 mM
Additional reagents: Lysozyme, Proteinase K
Preparation of Lysis Buffer for 10 Samples:
720 μl Tris buffer I
240 μL Lysis buffer I
96 u L RVC
Lysis
An overnight culture of E. coli (200 μL) has been centrifuged in Eppendorf tubes and the supernatant discarded.
Lysis buffer just before starting has been supplemented with Lysozyms (3 mg/mL) and added to the bacterial pellet (90 μL per pellet obtained from 200 μl culture). After suspension by ambient temperature 20 μL Proteinase K (1 mg/mL) have been added and the mixture was held for 10-200 min by temperature 20° C.-50° C. After this treatment the lysates were purified by two sequential simple spin column steps: Nexttec™ clean column and a spin column packed with the sorbent of Example 1. In both cases the spin columns were equilibrated before use following the recommendations for the Nexttec™ clean column of the producer (www.nexttec.biz). After equilibration the lysat was loaded onto the column and after a short centrifugation following again the recommendation for the Nexttec™ clean column of the producer the eluat was collected and analysed by gel electrophoresis (1% agarose in TAE buffer).
The results of RNA preparations are shown in the following picture. Obviously, than longer the incubation time and than higher the incubation temperature, than better yield of RNA wild be obtained.
The liver tissue was frozen overnight at −20° C.
The comparative analysed lysis procedures differed in the composition of the lysis buffers and the incubation time. The lysis incubation was at 37° C. All other conditions were as described under Example 3.
Gel electrophoresis of prepared RNA.
1 kb—DNA length standard, 0.1% DEPC
1, 7—lysis with VRC and 0.1% DEPC,
2, 8—lysis with VRC, RNasin and 0.1% DEPC
3, 9—lysis with VRC and Proteinase K
4,10—lysis with VRC, RNasin and Proteinase K
5,11—lysis with VRC, 0.1% DEPC and Proteinase K
6,12—lysis with VRC, 0.1% DEPC, Proteinase K and RNasin.
As it can be deduced from
Comparing the different variants of lysis buffer composition, the best conditions are the combination of the vanadyl-ribosyl complex with diethyl pyrocarbonat.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05108876.3 | Sep 2005 | EP | regional |
