WO1998014460A1

WO1998014460A1 - A method of separating a two phase system

Info

Publication number: WO1998014460A1
Application number: PCT/SE1997/001640
Authority: WO
Inventors: Pierre SÉVIGNY
Original assignee: Pharmacia Biotech AB; Amersham Pharmacia Biotech AB
Current assignee: Cytiva Sweden AB
Priority date: 1996-10-02
Filing date: 1997-09-30
Publication date: 1998-04-09
Anticipated expiration: 1999-04-02
Also published as: EP0931089A1; SE9603599D0; JP2001501821A

Abstract

A method of separating an upper aqueous phase from a lower organic phase in a two phase system. The method comprises the following steps: a) adding wax to the two phase system, b) heating the two phase system from a), whereby the wax is melted in the organic phase, c) cooling the two phase system from b) to solidify the wax-organic phase, d) withdrawing the upper aqueous phase. Further, the invention comprises a method of isolating RNA from cells by phenol-chloroform extraction in which an upper aqueous phase and a lower phenol-chloroform phase is formed, the aqueous phase containing the RNA. The method comprises the following steps: a) adding a solid way to the extraction mixture of cells, phenol and chloroform, b) heating the mixture to melt the wax, whereby the wax is mixed in the phenol-chloroform phase, c) cooling the mixture to solidify the wax-phenol-chloroform phase, d) withdrawing the upper aqueous phase containing the RNA.

Description

A method of separating a two phase system The present invention relates to a new method of separating an upper aqueous phase from a lower organic phase in a two phase system. The invention also discloses a method of isolating RNA from cells and a kit for the method. More precisely the invention discloses an extraction method by which wax is melted in the organic phase, and after cooling solidifies the organic phase. For RNA isolation the organic phase is a phenol-chloroform phase. RNA is used in different context to study genes and gene expression. RNA is a very unstable compound, partly because of its function in the cell, being a transitional form between different stages. RNA is easily degradable by RNAses,i.e. RNA degrading enzymes. This fact constitute the basic problem with RNA production as RNAses are omnipresent . An efficient method of RNA purification must therefore be rapid, minimize RNA degradation and generate RNA with a high degree of purity.

RNA is found in the cytosol of the cell. A common method to obtain the RNA from the cell is to mechanically decompose the cell tissue and dissolve the cellular membranes with phenol, whereby free RNA and DNA is achieved. Chloroform is used to extract the phenol constituent, forming two well separated phases, one upper aqueous phase con- taining RNA and one lower phenol-chloroform phase. Proteins and DNA are obtained at the interface between the aqueous and the organic phase and also in the organic phase . The aqueous phase is then withdrawn very carefully, e.g. by sucking, to leave behind all material in the other phases. The RNA is precipitated from the aqueous phase and washed. Purification of RNA with hot phenol is a method used since long time. This method is known to provide the highest level of RNA purity.

Other extraction methods are known as well . According to P. Chomczynski and N. Sacchi (Analytical Biochemistry 162, 156-159 (1987)) extraction with guanidinium thiocy- anate as well as a combination of guanidinium thiocyanate and phenol-chloroform is known. Also in these methods the aqueous phase has to be carefully separated to avoid contamination.

There are several draw backs with these methods . They are not rapid enough to enable processing many samples in parallel. The separation of the RNA containing aqueous phase is too slow. If many samples are processed simultaneously the RNA of the first samples will be destroyed at the time when the aqueous phase of the last sample is separated. It is difficult to remove the aqueous phase from the two phase system without contamination from the other phase. The RNA obtained will consequently be of low quality. Besides, the organic phase may expose the operator for hazardous chemicals.

The object of the present invention was to obtain a convenient method of separating the aqueous phase from the organic phase in a two phase system.

A further object of the present invention was to obtain an improved method of isolating RNA from cells or tissue by phenol-chloroform extraction without the draw backs of known methods .

Yet a further object of the invention was to present a kit for isolating RNA.

The objects of the invention are achieved by the methods and kit as claimed in the claims. According to the in- vention a method of separating an upper aqueous phase from a lower organic phase in a two phase system is obtained. The method is characterized by the following steps: a) adding wax to the two phase system, b) heating the two phase system from a) , whereby the wax is melted in the organic phase, c) cooling the two phase system from b) to solidify the wax-organic phase, d) withdrawing the upper aqueous phase.

According to a further aspect of the invention a method of isolating RNA from cells by phenol-chloroform extraction is obtained in which an upper aqueous phase and a lower phenol -chloroform phase is formed. The aqueous phase con- tains the RNA. The method is characterized by the following steps : a) adding a solid wax to the extraction mixture of cells, phenol and chloroform, b) heating the mixture to melt the wax, whereby the wax is mixed in the phenol-chloroform phase, c) cooling the mixture to solidify the wax-phenol- chloroform phase, d) withdrawing the upper aqueous phase containing the RNA.

According to yet a further aspect of the invention a kit for isolating RNA from cells by phenol-chloroform extraction is obtained. The kit comprises: a) wax, b) phenol extraction solution.

With the present invention it was found that an efficient, safe and easy way to separate the aqueous phase from the organic phase is achieved. As mentioned above, in the two phase system the organic phenol-chloroform phase has the highest density and will form the lower phase and the aqueous phase will form the upper phase. When wax with a lower density than water is used the wax will float on the water surface. However, when the mixture of the two phase system and wax is heated the wax will melt and mix in the organic phase. A wax with a higher density than water will of course be in the organic phase from the beginning and melt in it when heated. After cooling the wax-organic phase will solidify and form a solid plug in the bottom of the reaction vessel due to the higher density of the organic phase. In this manner the organic phase containing all cell constituents (proteins, lipids, etc.) susceptible to contaminate and affect the extracted nucleic acids, form a solid phase in the bottom of the reaction vessel. The aqueous phase containing the RNA can then easily be withdrawn without difficulties. The hazardous chemicals in the organic phase is now " trapped'' in a solid plug minimizing operator exposure to chemical solvents . As the aqueous phase can be quickly withdrawn in a convenient manner, many samples can be handled in a short time. Consequently, many samples can then be run in parallel. The quality of the RNA produced is also improved as the contaminants will stay firm in the solid plug. The heating to melt the wax is ad- vantageous for the quality of the sample because of denaturing of RNAses .

The method can also be used to isolate cellular DNA from the cell.

The method is easy to carry out . The sample which can be e.g. cell-culture, soft or hard tissue, such as tumour tissue, or full blood is mixed with phenol and chloroform and buffer according to any standard protocol . Wax is added, suitably in the form of pearls, pellets or powder. Different types of waxes can be used but it is important that the wax is not contaminated with any substance which disturbs the RNA stability, such as RNAses or is contaminated with compounds which may complicate down stream analysis. Preferred waxes are mineral waxes, such as petroleum waxes, or synthetic waxes such as ethylenic polymers and polyol ether-esters. These waxes are preferred to animal or plant waxes which may be contaminated by nature . Generally, waxes with a melting point between about 40 -

80°C can be used. Preferably the melting point lies within

45 - 65°C. The mixture of wax and sample is stirred and heated. The sample is mechanically decomposed in a conventional manner, such as by homogenization, to release the nucleic acids. Preferably the homogenization is made by using small glass beads (~ 2mm) which are added to a test tube. When the tube is vigorously shaken the decomposition of the cell material will be facilitated. After stirring the glass beads will sediment to the bottom of the tube because of a higher density and they will not interfere with plug formation. The heating temperature is adapted to the melting point of the wax to make sure that the wax melts during the heating step and mixes in the organic phase. The sample mixture is then cooled and because the organic phase is heavier than the aqueous phase the wax will solidify in the bottom of the sample vessel. Preferably the cooling is made by placing the samples on ice to bring about a quick and firm solidification of the wax. The aqueous solution containing RNA is withdrawn, e.g. by simply pouring it out of the vessel and the RNA is treated with ordinary ethanol precipitation or washing steps.

The amount of wax needed can easily be determined by the skilled man. It is important that the amount is enough to get a solid dense border to the aqueous phase. As a guideline an amount of 35 - 135 mg/ml extraction solution can be mentioned.

The method according to the invention is useful in combination with known phenol-chloroform extraction protocols. Preferably the extraction method which combines phenol - chloroform with a guanidinium compound is used. The use of acidic buffer in combination with phenol -chloroform is also good. The guanidinium method has been shown to result in a high yield of RNA and the RNA is both pure and undegraded. Also the kit according to the invention preferably com- prises the guanidinium compound or acidic buffer. The preferred guanidinium compound is guanidinium thiocyanate .

The invention will now be illustrated by the following examples which however are not intended to limit the invention. With parts and percent are meant parts by weight and percent by weight if not otherwise stated.

Example :

Extraction solution: 4 M guanidinium thiocyanate, 0.2 M sodium acetate pH=4.0, 0.5 % sarcosyl, 0.1 M 2- mercaptoethanol, 0.2 ml phenol/ml, 0.4 ml chloroform/ml and 8-10 glass beads (~ 2 mm in diameter) .

Sample: Hum B-cells, 0.5 10 cells/extraction tube. Wax: A: A mineral wax with a melting point 51-53°C (from the firm BDH Laboratory Supplies Poole, England, article no. 298395G) . B: AmpliWax™ from Perkin-Elmer with a melting point 55-58°C. An extraction tube is provided with 1.0 ml extraction solution and 75 mg wax (in the form of beads) , for wax A and 37 mg for wax B. The samples are transferred to the extraction tubes. The tubes are first shaken and then incu- bated at 65°C for 3 minutes (make sure that the wax is melted) . Then the tubes are vortexed for 10-15 sec, followed by incubation at 65°C for 2 minutes and incubation on ice at least for 1 in . The cooled aqueous phase containing RNA is precipitated by mixing with 0.4 ml of isopropanol. The precipitate is washed either by repeated centrifugation and aspiration in 70 % methanol using an Eppendorf centrifuge, or alternatively, by adding the precipitate to a filtration device like the Pharmacia EasyPrep® using glass filters or the EasyPrep® Blue filters device (washing wells) followed by repeated washing of the precipitate using 70% ethanol .

Finally, in both these alternative procedures the washed RNA is dissolved for 3 minutes using 50 μl DEPC- treated (diethylpyrocarbonate) water containing 25 U RNA- guard® (a ribonuclease inhibitor isolated from human placenta, from Pharmacia Biotech) . The RNA is collected by pipetting or, in the case of EasyPrep, by applying low pressure .

The quality of the RNA is evaluated by converting a small portion of the preparation to cDNA and further amplification by PCR. cDNA reaction:

The RNA samples are heat denatured at 65°C for 3 minutes and is then quenched on ice. 25μl cDNA-mix, 20μl MMULV RT (Moloney Murine leukemia virus reverse transcriptase) , 5μl RNAguard and 50μl RNA sample are mixed in reaction tubes and incubated at 37°C for 1 min. The cDNA samples are stored at - 20°C. cDNA-mix: 90mM Tris-HCl pH 8.3 138mM KCl

18 πiM MgCl₂ 30mM DTT

3.6mM dATP, dCTP, dTTP, dITP and 0.9mM dGTP

0.152 A260 U pd(N)g (random hexamer)

PCR reaction on as an example a 482 bp out of the p53 cDNA.

A Perkin Elmer 9600 PCR machine is used.

In 0.2 ml reaction tubes are mixed:

5μl PCR II buffer (lOx) (Perkin Elmer) 5 pmol primer (PCR primer for p53, fragment 3)* 5 pmol primer (PCR primer for p53, fragment 3)* 5μl cDNA

1.2μl 25mM MgCl2

O.'δμl AmpliTaq™ (Taq polymerase from Perkin Elmer) (4U) and water up to 50μl. The sample are cycled 38x with the AUTO profile: 94°C 15sec, 58°C 30sec, 72°C 45sek, and end with a 5min. hold at

72°C. Subsequent quenching to 4°C.

The amount of PCR product obtained is : Sample with wax A: 22 ng/μl, Sample with wax B: 10 ng/μl.

The method gave good products, is easy and fast to use and is safe for the user.

* PCR primer for p53 , fragment 3) : 5'-TGG CCC CTC CTC AGC ATC TTA-3' and B-5'-CAA GGC CTC ATT CAG CTC TC- 3'(B=biotin labelled)

Claims

1. A method of separating an upper aqueous phase from a lower organic phase in a two phase system, characterized by the following steps: a) adding wax to the two phase system, b) heating the two phase system from a) , whereby the wax is melted in the organic phase, c) cooling the two phase system from b) to solidify the wax-organic phase, d) withdrawing the upper aqueous phase.

2. A method of isolating RNA from cells by phenol- chloroform extraction in which an upper aqueous phase and a lower phenol-chloroform phase is formed, the aqueous phase containing the RNA, characterized by the following steps: a) adding a solid wax to the extraction mixture of cells, phenol and chloroform, b) heating the mixture to melt the wax, whereby the wax is mixed in the phenol-chloroform phase, c) cooling the mixture to solidify the wax-phenol- chloroform phase, d) withdrawing the upper aqueous phase containing the RNA.

3. A method according to claim 1 and 2, characterized in that the wax is a mineral wax.

4. A method according to claim 2, characterized in that the extraction mixture further comprises a guanidinium compound .

5. A method according to claim 4, characterized in that the guanidinium compound is guanidinium thiocyanate.

6. A kit for isolating RNA from cells by phenol- chloroform extraction, said kit comprising: a) wax, b) phenol extraction solution.

7. A kit according to claim 6, characterized in that the kit further comprises glass beads.

8. A kit according to claim 6 or 7, characterized in that the extraction solution further comprises a guanidinium compound.

9. A kit according to claim 8, characterized in that the guanidinium compound is guanidinium thiocyanate.

10. A kit according to claim 6 or 7, characterized in that the extraction solution further comprises acidic buffer.

11. A kit according to any of the claims 6-10, characterized in that the wax is a mineral wax.