WO2004079333A2

WO2004079333A2 - Method and kit for extracting rna from sputum

Info

Publication number: WO2004079333A2
Application number: PCT/KR2004/000053
Authority: WO
Inventors: Chang-Ho Jeon; Jong-Wook Park
Original assignee: Ic & G Co., Ltd.
Priority date: 2003-01-15
Filing date: 2004-01-14
Publication date: 2004-09-16
Also published as: WO2004079333A3

Abstract

Disclosed are a method and kit for extracting RNAs from a sputum sample. The method comprises the steps of mixing sputum with guanidinium thiocyanate buffer so that the sputum can be liquefied and cell-lysed, and separating RNAs from the liquefied and cell-lysed sputum. And the kit comprises guanidinium thiocyanate buffer.

Description

METHOD AND KIT FOR EXTRACTING RNA FROM SPUTUM

Field of the Invention The present invention relates to a method and kit for extracting RNAs from a sputum sample.

Background of the Invention

It is generally known that RNAs are not easily extracted from a sputum sample without damage since a sputum sample is mixed with a large quantity of normal cells, varieties of bacteria, proteins, mucilage, polysaccharides and the like. Because a sputum sample particularly contains many bacteria, even slight damage of cells easily leads to the degradation of the RNAs. Therefore, it is difficult to clinically apply a conventional polymerization chain reaction to a sputum sample. Meanwhile, a method for extracting RNAs from a sputum sample without damage necessarily needs to be developed, in order to detect clinically significant oncogenes of cancerous cells, present in a small quantity in a sputum sample.

A conventional method for extracting RNAs from a sputum sample passes through the steps of completely liquefying a sputum sample using dithiothreitol or N- acetyl-L-cysteine, accumulating cells by centrifuging the resultant mixture, lysing the accumulated cells, and separating RNAs from the lysed cells (hereinafter referred to as the "DTT method")(Ann Intern Med 1998; 109:7-10. J Virological Methods 2001; 94: 129-135). However, the conventional method requires 15 minutes or 1 hour to completely liquefy a sputum sample, and is complex, and shows low extraction efficiency.

The present invention has been made keeping in mind the above-described problems in the conventional art, and therefore the present invention provides a method and kit for extracting RNAs from a sputum sample, which may quickly and easily separate RNAs from a sputum sample with high extraction efficiency.

Brief Description of the Accompanying Drawing

Fig 1 schematically shows a process for extracting RNAs from a sputum sample, according to one embodiment of the present invention.

Fig 2 schematically shows a process of extracting RNAs from a sputum sample, according to another embodiment of the present invention.

Fig 3 shows a result of electrophoresis of MA (melanoma antigen) gene for comparing the extraction efficiency of the extracting method according to the present invention and that of the extracting method according to the conventional method. Fig 4 shows a result of electrophoresis of GAPD (glyceraldehydes phosphate dehydrogenase) gene for comparing the extraction efficiency of the extracting method according to the present invention and that of the extracting method according to the conventional method.

Detailed Description of the Invention

An object of the present invention is to provide a method and kit for extracting RNAs from a sputum sample, which may quickly and easily separate RNAs from a sputum sample with high extraction efficiency.

In an aspect, the present invention provides a method for extracting RNAs from a sputum sample.

The extracting method of the present invention, particularly, comprises the following steps of:

(a) mixing a sputum sample with guanidinium thiocyanate buffer (hereinafter referred to as the "GT buffer") so that the sputum sample can be liquefied and cell-lysed; and

(b) separating RNAs from the liquefied and cell-lysed sputum sample. According to the above-mentioned extracting method of the present invention, it is possible to quickly and easily extract RNAs from a sputum sample with comparatively high extraction efficiency. In the extracting method of the present invention, the GT buffer used in the above step (a) may comprise various additives in addition to guanidinium thiocyanate. As such additives, there may be exemplified, for example, Tris-Cl, β-mercaptoethanol, dithiothreitol and the like. As such additives, t-octylphenoxypolyethoxyethanol (comes into market under the trademark of "Triton-X-100") is also exemplified. However, if any buffer contains guanidinium thiocyanate, such buffer is sufficient to be used as the GT buffer of the present invention, irrespective of density of guanidinium thiocyanate, existence and density of such additives. Therefore, the GT buffer is used as a general term meaning all kinds of buffers in this specification including the accompanying claims insofar as the buffers comprise guanidinium thiocyanate. However, in view of RNA extraction efficiency, the GT buffer preferably comprises 3 M or more guanidinium thiocyanate, and more preferably, comprise 4 M or more guanidinium thiocyanate. In the same view of RNA extraction efficiency, the still more preferable GT buffer comprises 4.5 M or more guanidinium thiocyanate, and, as additives, 0.1 M or more Tris-Cl, 1% or more β-mercaptoethanol, and 0.1% or more dithiothreitol. Continuing the still same view of RNA extraction efficiency, the still more preferable GT buffer comprises 4.8 M or more guanidinium thiocyanate, and, as additives, 0.1 M or more Tris-Cl, 1% or more β-mercaptoethanol, and 0.1% or more dithiothreitol. In addition, all the above preferable GT buffers may comprise t- octylphenoxypolyethoxyethanol as the additives, preferably, in the amount of 1% or more. The most preferable GT buffer comprises 5.0 M guanidinium thiocyanate, and, as additives, 0.1M Tris-Cl, 1% β-mercaptoethanol, and 0.1% dithiothreitol.

All the GT buffers, which are mentioned to be preferable in view of RNA extraction efficiency in the above, has been obtained through repeated experiments of the present inventor(s) experiencing several occurrences of trial and error. Although the most preferable GT buffer, mentioned in the above, was used in the example of this invention, it should be understood that the GT buffer was used since it is most preferred among the above-mentioned GT buffers.

In the extracting method of the present invention, the above step (a), in which a sputum sample is mixed with GT buffer, allows the sputum sample to be liquefied and cell-lysed through mixing with GT buffer. If the sputum sample is liquefied and cell- lysed, the procedure to extract RNAs from the liquefied and cell-lysed sputum sample is very easy and may be performed using procedure well-known to those ordinarily skilled in the art. To explain such procedure with a general example, nucleic acid binding beads are added to the liquefied and cell-lysed sputum sample, resulting in complexes of nucleic acids and beads, and then nucleic acids are separated from the complexes, and finally RNAs are separated from the nucleic acids using suitable reagents.

Therefore, in the present invention, the above step (b), in which RNAs is separated from the sputum sample liquefied and cell-lysed in the above step (a), is preferred to comprise the steps of (i) adding nucleic acid binding beads to the liquefied and cell-lysed sputum sample so that complexes of nucleic acids and beads can be formed, (ii) separating the nucleic acids from the complexes, and (iii) separating RNAs from the separated nucleic acids.

In preferable embodiment of the present invention, the above step (ii) for separating the nucleic acids from the complexes is preferred to be performed using a phenol-guanidinium thiocyanate reagent (hereinafter is referred to as the "PGI reagent"). The PGI reagent allows nucleic acids to be easily eluted from the complexes. Any PGI reagents are sufficient to be used in the present invention insofar as the PGI reagents comprise phenol and guanidinium thiocyanate as effective agents. The PGI reagent may further comprise t-octylphenoxypolyethoxyethanol, N-acetyl-L-cysteine and the like, as additives. In view of nucleic acid elution efficiency, the above PGI reagent is preferred to comprise 0.5 M or more phenol and 0.5 M or more guanidinium thiocyanate. In the same view of nucleic acid elution efficiency, the PGI reagent may further comprise 0.1 M or more - t-octylphenoxypolyethoxyethanol and 0.1 M or more N-acetyl-L-cysteine as additives in addition to the above-mentioned phenol and guanidinium thiocyanate. PGI reagent having a trademark of "Trizol" (available from Life Technologies, Inc.), which comprises phenol and guanidinium thiocyanate as effective agents, was used in the example of the present invention. In another preferable embodiment of the present invention, the above step (b) may further comprise the step of eliminating non-specific bonds between impurities and nucleic acid binding beads using suitable washing buffer so that the impurities can be separated from the nucleic acid binding beads, next to step (i). This step ultimately enables the RNA extraction efficiency to be heightened. The suitable washing buffer to be used at the above step is easily prepared by those ordinarily skilled in the art. The washing buffer used in the example of the present invention was prepared, after preparing the buffer containing 100 mM NaCl and 10 mM Tris-HCl (pH 7.5), by adding fourfold volume of ethanol to the buffer so that it could contain 20 mM NaCl and 2 mM HC1 (pH 7.5) at the final density. In still another preferable embodiment of the extracting method of the present invention, the above step (b) is preferred to comprise the steps of (i) adding mRNA binding beads to the liquefied and cell-lysed sputum sample so that complexes of mRNAs and beads can be formed, and (ii) separating the mRNAs from the complexes. If the present invention further comprise such a step, it would be possible to easily separate clinically significant mRNAs. In this case, the mRNA binding bead is preferred to be a bead bound with biotinylated oligo (dT) and more preferred to a streptavidin bead bound with biotinylated oligo (dT). Any mRNA binding beads can be used insofar as they have the ability to capture mRNA.

In another aspect, the present invention provides a kit for extracting RNAs from a sputum sample, comprising GT buffer.

The GT buffer contained in the extracting kit enables a sputum sample to be liquefied and cell-lysed, as already described in the above extracting method of the present invention. If the sputum sample is liquefied and cell-lysed, RNAs may be easily extracted from the liquefied and cell-lysed sputum sample using procedures well-known to those ordinarily skilled in the art or the above-described extracting method.

The above description related to the GT buffer used at the above extracting method of the present invention may apply to the GT buffer contained in the extracting kit, specifically in relation with the composition and density of the components and the like. Therefore, although the composition and density of the components and the like are not described in detail herein, the above description related to the GT buffer used at the above extracting method should be deemed to be applied to the GT buffer contained in the extracting kit with necessary modifications.

Meanwhile, the extracting kit may further comprise nucleic acid binding beads as one of means for extracting RNAs. At this case, if the nucleic acid binding beads are mRAN binding beads, it is possible to quickly and easily separate clinically significant mRNAs. Therefore, the nucleic acid is preferred to be mRNA. The mRNA binding bead is preferred to be a bead bound with biotinylated oligo (dT) and more preferred to be a streptavidin bead bound with biotinylated oligo (dT). Other mRNA binding beads may be used in the extracting kit of the present invention.

Further, the extracting kit of the present invention may further comprise PGI reagent in addition to the above GT buffer and nucleic acid binding beads. The above description related to the PGI reagent used at the above extracting method of the present invention may apply to the PGI reagent contained in the extracting kit, specifically in relation with the composition and density of the component and the like.

Furthermore, the extracting kit of the present invention may further comprise washing buffer. The washing buffer enables impurities to be separated from the nucleic acids binding beads by eliminating non-specific bonds between impurities and nucleic acid binding beads, resulting in ultimately heightening the RNAs extraction efficiency, as described in the above extracting method of the present invention.

Description of the Preferred Embodiment

Hereinafter, the present invention will be described in detail with reference to the following examples and comparative examples, which should not be construed to be limited to the scope of the present invention.

Example 1 : Extraction of RNAs from a sputum sample

Example 1-1 : Description of reagents used in this example

GT Buffer: The GT buffer comprises 5.0 M guanidinium thiocyanate, 0.1M Tris- Cl, 1% β-mercaptoethanol, and 0.1% dithiothreitol. The GT buffer has an ability to liquefy a sputum sample and lyse cells present in a sputum sample and stabilize RNAs. The GT buffer may comprise t-octylphenoxypolyethoxyethanol (Triton-XlOO). Nucleic Acid Binding Bead (NA bead): Nucleic acid binding bead is made of polystyrene, and contains capsulated oxidized steels therein. The nucleic acid binding bead also comprises - COOH groups on its external surface. The -COOH groups on the external surface of the NA bead bind to RNAs present in a liquefied and cell-lysed sputum sample. The capsulated oxidized steels in the interior of the bead allow the bead to be under magnetic attraction, and thus enabling RNAs bound to the beads to be gathered. Such functions of the NA bead allow the NA bead to be suitably used for the purpose of extracting RNAs from a sputum sample.

Washing Buffer: Washing buffer is manufactured, after manufacturing the buffer containing 100 mM NaCl and 10 Mm Tris-HCl (pH7.5), by adding fourfold volume of cold ethanol to the manufactured buffer so that the resultant buffer can contain 20 mM NaCl and 2mM HC1 (pH 7.5) at the final density. The washing buffer has an ability to eliminate non-specific binding, and thus allowing impurities to be separated from the NA beads. Therefore, it is possible to wash off impurities non-specifically bound with the NA beads using the washing buffer.

PGI Reagent: PGI reagent is referred to as Trizol in the trademark (available from Gibco BRL Life Technologies in U.S.A.). The PGI reagent has an ability to suppress ribonuclease activity, and thus enabling RNAs, DNAs and proteins to be separated at one time and allows RNAs to be stabilized. In addition, the PGI reagent allows nucleic acids bound with the NA beads to be eluted into a solution.

Chloroform: Chloroform binds to proteins, etc. to form a precipitate. Therefore, the chloroform is used in separating RNAs from DNAs and proteins. Isopropyl Alcohol: Isopropyl alcohol has an ability to condense RNAs by precipitating the separated RNAs.

RNase-Free 75% Ethanol: RNase-free 75% ethanol is used in washing off impurities present in the precipitated RNAs.

Diethylpyrocarbonate Distilled Water: Diethylpyrocarbonate distilled water is used in dissolving the precipitated RNAs.

Example 1-2. Extraction process of RNAs from a sputum sample

First, an extraction process of RNAs from a sputum sample is schematically described below with reference to Fig 1.

First of all, a sputum sample was prepared (1), and then GT buffer was added to the prepared sample (2) to form a mixture (3). NA beads were added to the mixture (4) to be mixed (5). The resultant supernatant was removed (6) and the NA beads were washed off (7). PGI reagent was added to and mixed with the NA beads for 10 minutes at 70 ^°C so that nucleic acids bound to the NA beads could be eluted (8, 9). The eluted nucleic acids were transferred to a fresh tube (10), into which chloroform was added. After the tube was centrifuged, the supernatant containing RNAs was recovered (11). Isopropyl alcohol was added to the recovered supernatant in order to precipitate RNAs (12). The supernatant was removed and 76% ethanol was added to the precipitate to wash off (13). The resultant mixture was centrifuged, and then the supernatant ethanol was removed. Finally, distilled water was added to the remnants so that RNAs could be dissolved (14).

Hereinafter, an extraction process of RNAs from a sputum sample is described with reference to a preferable embodiment.

(1) Liquefaction and cell-lysis of a sputum sample by using GT buffer A sputum sample was obtained from a patient. GT buffer was added to the sputum sample soon after it was obtained. The resultant mixture was vortexed until the sputum sample became liquefied and cell-lysed completely. At this point, GT buffer can be further added in case that the sputum sample is not completely liquefied and cell-lysed. The liquefied and cell-lysed mixture can be maintained stably for about 6 months by keeping it at -20 ^°C to -70 ^°C . This step is to liquefy mucilage and lyse cells in the sputum sample, whereby the exposed RNAs are stabilized. Therefore, this step enables the sputum sample to be quickly treated and also enables the exposed RNAs to be stabilized.

(2) Addition of NA beads

Homogenized NA beads were added to the liquefied and cell-lysed sputum sample. The resultant mixture was vortexed. The vortexed mixture was stored at room temperature in a roller mixer. At this point, it is preferable to prepare a washing buffer by adding cold ethanol to nucleic acid buffer.

(3) Removal of supernatant The liquefied and cell-lysed mixture was centrifuged at 2000 rpm for 5 minutes.

After centrifugation, the supernatant was removed, and the NA beads were suspended and transferred to a 1.5 ml tube. The remaining NA beads were recovered with a 50 ml tube containing 0.5 ml of washing buffer, and then the recovered NA beads were transferred into the 1.5 ml tube.

(4) Washing of NA beads

© The tube containing NA beads was placed into a magnetic separator so that the NA beads could be under magnetic attraction, and then the NA beads were recovered by removing the supernatant. © 1 ml of washing buffer was added into the tube, and then the recovered NA beads were washed by carefully mixing with a pipette. After the tube was placed into a magnetic separator for the NA beads to be under magnetic attraction, the buffer contained in the tube was removed with a pipette (repeated three times). At the final third time, the buffer was removed once, and after a while, the remaining buffer was completely removed.

(5) Elution of nucleic acids

© NA beads were re-suspended by adding 800 ^ of PGI reagent into the tube containing the NA beads. By adding PGI reagent to NA beads, this step was performed in combination of NA bead method and Trizol method. RT-PCR is not easily performed using RNAs extracted using GT buffer, since a sputum sample is generally mixed with mucilage, polysaccharides and the like. Therefore, after washing RNAs extracted using GT buffer, this step is necessary for easily performing RT-PCR by removing proteins, polysaccharides and the like from the washed RNAs with Trizol solution once more. © The resultant tube was stirred at 1400 rpm in a shaker (Eppendorf Shaker) at 70 ^°C for 10 minutes. At this point, if there is no shaker, the tube may be vortexed twice at an interval of 3 minutes in a dry bath at 70 °C .

(I) The tube was placed into a magnetic separator so that the NA beads contained in the tube could be under magnetic attraction. The eluted nucleic acids were transferred to a fresh tube. At this point, it is necessary to be careful so that the NA beads cannot be transferred to the tube along with the eluted nucleic acids.

® After cooling the tube at room temperature, the tube was sealed and shaken vigorously for 15 seconds. And then, the tube was centrifuged at 1200 rpm for 15 minutes.

As a result of centrifugation, the tube was divided into a red-colored lower layer containing chlorophenol, middle layer, and colorless aqueous upper layer containing

RNAs. The amount of the colorless aqueous upper layer commonly amounts to 60 %> of the quantity of the added Trizol. If not, the tube needs to be centrifuged for 5 to 10 minutes.

(6) Precipitation of RNAs

© The same amount of the isopropylalcohol is put into the tube. After the tube was placed at room temperature for 10 minutes, and then the tube was centrifuged at 12000 rpm at 4 ^°C for 10 minutes. At this point, RNAs precipitate can appear to be pellet like gel before centrifugation.

(7) Washing of RNAs

The resultant supernatant was removed from the centrifuged tube. After 1 ml of 75%o ethanol was added to the tube, the tube was mixed inversely and centrifuged at 12000 rpm at 4 °C for 5 minutes.

(8) Re-dissolution of RNAs

© The resultant supernatant was removed from the centrifuged tube. RNAs pellet was recovered and air-dried for 5 to 10 minutes (not be vacuum-dried). At this time, the pellet should not be completely dried. © 251& of RNase-free distilled water was carefully mixed with the dried RNAs pellet. The resultant mixture was placed at room temperature for 10 minutes and then placed immediately in ice.

(D After the tube containing RNAs was tapped 5 times, 11.0 of RNAs was taken out from the tube. Reverse transcription reaction was performed using the RNAs. The remaining RNAs were stored in a refrigerator at 70 °C (for re-testing).

The sputum sample used in this example was obtained from a patient in hospital.

Example 2: Separation of mRNAs from a sputum sample

Example 2-1: Description of reagents used in this example GT Buffer: The same buffer as mentioned in the Example 1 was used in this example.

Biotinylated oligo (dT): The biotinylated oligo (dT) was prepared by binding biotin to oligo (dT). In this Example, 10 pmol/ β biotinylated oligo (dT) was used in an amount of 20 βi. .

Streptavidin Bead (SA bead): Streptavidin in the Streptavidin bead allows the beads to be under magnetic attraction. 10 mg/ml of SA beads were used herein in an amount of 50 ^2 per each sample.

Washing Buffer 1: Washing buffer 1 is manufactured, after manufacturing the buffer containing 100 mM NaCl and 10 mM Tris-HCl (pH7.5), by adding fourfold volume of ethanol to the manufactured buffer so that the resultant buffer can contain

20 mM NaCl and 2mM HC1 (pH 7.5) at the final density. This buffer has an ability to eliminate non-specific binding, and thus allowing impurities to be separated from the SA beads. Therefore, it is possible to wash off impurities non-specifically bound with the S A beads using the washing buffer 1.

Washing Buffer 2: Washing Buffer 2 contains 1 mM EDTA, 1M NaCl, and 10 mM Tris-HCl (pH 7.5).

Washing Buffer 3: Washing Buffer 3 contains 20 mM NaCl and 2 mM Tris-HCl (pH 7.5).

Diethylpyrocarbonate distilled water (Diethylpyrocarbonate-DW): Diethylpyrocarbonate distilled water is used to dissolve the extracted RNAs.

Example 2-2: Process of separating mRNAs from a sputum sample

As schematically shown in Fig 2, a sputum sample was prepared (1), and GT buffer was added to the prepared sputum sample (2), and then the resultant mixture was stirred (3). SA beads bound with biotinylated oligo (dT) were added to the stirred mixture (4) to form a mixture (5). After the supernatant was removed from the mixture (6), the SA beads were washed off (7), followed by adding distilled water (9) and mixing for 3 minutes at 70 °C, whereby mRNAs were separated(lθ).

Hereinafter, an extraction process of mRNAs from a sputum sample is described with reference to a preferable embodiment.

(1) Liquefaction and a cell-lysis of a sputum sample by using GT buffer This process was carried out by the same procedure as described in the process 1-1 of the Example 1.

(2) Binding biotinylated oligo (dT) to S A beads φ SA beads were completely suspended and added into a tube in an amount of 50 # per one sample. A supernatant (containing S A beads) was recovered by placing the tube into a magnetic separator so that S A beads could be under magnetic attraction. (B) After 150 μJl of distilled water and 20 μi of biotinylated oligo (dT) were added to the tube to be completely mixed, the resultant was placed at 37 °C for 3 minutes.

(3) Addition of S A beads bound with biotinylated oligo fdT)

170/^6 of SA beads bound with biotinylated oligo (dT) were added into the tube containing the mixture of a sputum sample and GT buffer, and then was mixed at room temperature for 30 minutes with roller mixer.

(4) Collection of S A beads φ A supernatant was removed by placing the mixed tube into a magnetic separator. At this point, the supernatant may be removed by centrifuging the mixed tube at 2,700 rpm for 10 minutes, if there is no magnetic separator. φ SA beads were suspended by adding 0.5 ml of the washing buffer 1 into the tube, and then were transferred into a 1.5 ml tube. (3) The remaining SA beads were recovered by adding 0.5 ml of the washing buffer 1 into the tube, and then were collected into the 1.5 ml tube.

(5) 1st Washing of SA beads φ A supernatant was removed by placing the 1.5 ml tube into a magnetic separator so that SA beads could be under magnetic attraction. φ 1 ml of the washing buffer 1 was added into the 1.5 ml tube, and then the beads were washed by carefully mixing with a pipette. The 1.5 ml tube was placed into a magnetic separator for the SA beads to be under magnetic attraction, and then the buffer contained in the tube was removed with a pipette. (3) This process was repeated once more.

® At the final third time, the buffer was removed once, and after a while, the remaining buffer was completely removed.

(6) 2nd Washing of SA beads φ SA beads were re-suspended by using 1.0 ml of the washing buffer 2. φ S A beads were attracted by a magnetic separator.

(3) A supernatant was removed.

® The resultant was washed with 1 ml of the washing buffer 3 using the same procedure as in the step (5).

(7) Elution of mRNAs from S A beads φ S A beads were re-suspended with 50 i£ of distilled water, and then they were placed at 70 °C for 3 minutes, thereby eluting mRNAs. φ After SA beads were separated by using a magnetic separator, mRNAs in a supernatant were collected and transferred into a fresh tube, and then the tube was immediately stored in a refrigerator (mRNAs may be collected by further adding distilled water if the density mRNAs is high. The reason is that mRNAs may not be easily collected because of its high viscosity). Comparative Example 1: Comparison of time required to liquefy a sputum sample and stabilize RNAs between the method of the present invention and conventional DTT method

Comparative Example 1-1 : Experimental methods

The sputum sample used in this example was obtained from patients in hospital requesting their sputum to be microorganism-cultivated. After cultivation, the remaining 20 sputum samples having seemingly similar viscosity were divided into two groups, each of which consists of 10 sputum samples. The time required to liquefy each sputum sample and stabilize RNAs was determined respectively for each group with the buffer, used in the DTT method, containing 0.3 % dithiothreitol and 0.02 M of EDTA (pH 7.0) and GT buffer used in the present invention.

(1) DTT method φ The dithiothreitol-EDTA buffer was added into a tube containing a sputum sample divided into the first group, in the same amount, and then stirred. φ The sputum sample contained in the tube was liquefied by using a vortexer. The dithiothreitol-EDTA buffer may be further added when the sputum sample is not completely liquefied. (3) After the tube was centrifuged at 2,000 rpm for 10 minutes, the resultant supernatant was removed, whereby cell layer was collected.

® The collected cell layer was suspended by adding 3 ml of PBS (phosphate buffered saline), and then was accumulated by centrifuging at the same rpm for 5 minutes. (5) The resultant supernatant was removed, thereby collecting cell layer. The collected cell layer was suspended again by adding 3 ml of PBS, and then the suspended cell layer was accumulated again by centrifuging under the same condition.

(6) After the supernatant was removed, the accumulated cell layer was re- suspended by adding of 1 ml of PGI reagent. Hereby, the process for extracting RNAs was completed. The time required was determined by measuring the time until then. ® This procedure was repeated for each of the other 9 tubes.

(2) Method of the present invention φ GT buffer was added into a tube containing a sputum sample divided into the second group, in the same amount, and then stirred. φ The sputum sample contained in the tube was liquefied by using a vortexer. The GT buffer may be further added when the sputum sample is not completely liquefied.

(H) When the sputum sample was liquefied, the process for extracting RNAs was completed. The time required was determined by measuring the time until then.

® This process was repeated for each of the other 9 test tubes.

Comparative Example 1-2: Results

(1) According to the present invention, it was possible to liquefy a sputum sample and stabilize RNAs within 5 minutes by adding twofold or threefold the volume of the GT buffer to a sputum sample and stirring the resultant mixture.

(2) According to the conventional method, 30-60 minutes were required from separation of cell layer to stabilization of RNAs. Moreover, even the complete liquefaction was impossible in some high viscous sputum samples.

Comparative Example 2: Comparison of RNA extraction efficiency between the method of the present invention and conventional DTT method

Comparative Example 2-1. Experimental method The RNA extraction efficiency for the sputum samples obtained in the

Comparative Example 1 was compared between the method of the present invention and the conventional DTT method. MA (melanoma antigen)-positive SNU 484 cells were added into 5 ml of each of sputum samples in numbers of 5, 20, 80 and 320, respectively, and RNAs were extracted from the sputum samples. RT-PCR of MA gene was performed using the extracted RNAs. After this procedure was repeated 10 times, the extraction efficiency for certain RNAs was compared between the method according to the present invention and the conventional DTT method by measuring the amount of MA gene detected.

fl) DTT method φ Fractionation of RNAs

(a) The cell layer of a sputum sample, to which Trizol was added, was placed at room temperature for 5 minutes.

(b) 0.2 ml of chloroform was added per 1 ml of Trizol. The mixture was sealed and vigorously stirred by hand for 15 seconds, and then was centrifuged at 12,000 rpm and 4 °C for 15 minutes.

(c) The mixture was fractioned into a red-colored lower layer containing chlorophenol, middle layer, and colorless aqueous upper layer. (d) The upper layer amount to 60% of the quantity of the added Trizol. φ Precipitation of RNAs

(a) A supernatant was transferred into a fresh tube. At this point, the remaining layer may be used for the extraction of DNA or proteins.

(b) The same amount of isopropyl alcohol (per 1 ml Trizol) was added into the tube and the tube was placed at room temperature for 10 minutes, followed by centrifuging at 12,000 rpm and 4 °C for 10 minutes. At this point, the centrifugation should be performed for the exact time of 10 minutes.

Φ Washing of RNAs

After removing the resultant supernatant, 1 ml of 75% ethanol was added and vortexed, followed by centrifuging at 7,500 rpm and 4 °C for 5 minutes.

® Re-dissolution of RNAs

(a) The resultant supernatant was removed and the resultant RNA pellet was air- dried for 5 to 10 minutes.

(b) 25 μi of RNAase-free water was added to the RNA pellet. The mixture was mixed with a pipette, and then was placed at 55 to 60 °C for 10 minutes.

(2) Method according to the present invention φ 150/z# of well-mixed NA beads was added to the liquefied sputum sample and the resultant sputum sample was vortexed. If GT buffer is further added, NA beads should be also additionally added in proportion to the amount of the further added GT buffer. φ The vortexed sputum sample was placed at a constant room temperature for 30 minutes in roller mixer. φ After the liquefied sputum sample was centrifuged at 2,000 rpm for 5 minutes and the resultant supernatant was removed, the beads were suspended and transferred into 1.5 ml tube. The remaining beads were recovered with the 50 ml tube, into which 0.5 ml of NA bead washing buffer was added, and transferred into the 1.5 ml tube.

® Washing of NA beads (a) After the tube containing the recovered beads was placed into the magnetic separator so that the NA beads could be under magnetic attraction, the resultant supernatant was removed.

(b) 1 ml of washing buffer was added into the tube, and then the recovered NA beads were washed by mixing with a pipette. After the tube was placed into the magnetic separator for the beads to be under magnetic attraction, the buffer was removed with a pipette (repeated three times). At the third time, the buffer was removed once, and after a while, the remaining buffer was completely removed. © Elution of Nucleic Acid (a) 800 £ of PGI reagent was added into the tube, and then NA bead was re- suspended.

(b) The tube was stirred at 1,400 rpm and 70 °C for 10 minutes with a shaker (Eppendorf shaker).

(c) The tube was placed into a magnetic separator for the beads to be under magnetic attraction, and the eluted nucleic acid was transferred into a fresh tube. At this point, it is necessary to be careful so that the NA beads cannot be transferred to the tube along with the eluted nucleic acids.

(d) After the tube was cooled at room temperature, 0.2 ml of chloroform was added into the tube. The resultant tube was sealed and vigorously stirred by hand for 15 seconds, and then was centrifuged at 12,000 rpm and 4 °C for 15 minutes.

(3) RT-PCR and nested PCR RT-PCR and nested PCR were commonly carried out for each of both RNA groups, extracted respectively using the conventional DTT method and the method of the present invention, as described below. φ The tube containing ll.O € of RNAs solution was placed at 70 °C for 10 minutes, and then were cooled with ice so that the second structure of RNAs could be eliminated. φ 20 of RNAs (2 βg) was added into the tube, and RT-PCR was carried out under the following conditions:

5X RT buffer (Promega, U.S.A.) 40

10 mM dNTP mixture 1 ø MMLV (200 VI ø) 0.5 id

RNase inlύbitor (40 Wø) 0.5 μi

Oligo dT primer (100 pmol) 1 ø

Distilled water 11 μβ RNA 20 A drop of mineral oil was added into the tube, and then RT reaction was carried out by placing the tube into a PCR apparatus (Perkin Elmer, Cetus 2400) (Conditions: 25 °C, 10 minutes; 42 °C, 60 minutes: 1 cycle).

® PCR-1 mixture was prepared as follows: 10X PCR buffer 30

25 mM MgCl₂ 2A0 lOO mM dNTP 0.3 ^

10 pmol primer C 1 0.1 ^€

10 pmol primer C2 0.1 ø PCR enzyme 1U 0.1^

Distilled water 22.0/^

© A drop of mineral oil was added into the tube, and then PCR was carried out by placing the tube into a PCR apparatus as follows (At this point, the reaction time should be adjusted appropriately according to a PCR apparatus, since 900 bp is generally synthesized in case of PE 2400).

94 °C, 10 minutes: 1 cycle (pre-treated at a constant temperature for Gold Taq (PE)) 94 °C, 30 seconds; 58 °C 30 seconds; 72 °C, 1 minute: 30 cycle

72 °C, 3 minutes: 1 cycle

(7) PCR-2 mixture was prepared as follows:

1 OX PCR buffer 30

25 mM MgCl₂ 2A/d lOO mM dNTP 0 30

10 pmol primer C3 0 10

10 pmol primer C4 0 lø

PCR enzyme 1U 0 10

Distilled water 22.0/^ ® 20 of 1 st PCR product was added to the above mixture.

(9) The resultant mixture was placed into the PCR apparatus, and nested PCR was carried out (490 bp was amplified within MA gene) as follows:

94 °C, 10 minutes: 1 cycle (pre-treated at a constant temperature for Gold Taq (PE))

94 °C, 30 seconds; 60 °C, 30 seconds; 72 °C, 45 seconds: 30 cycles

72 °C, 3 minutes: 1 cycle

Primer sequences and the sizes of PCR products used in the electrophoresis are given in the following Table 1.

[Table 1]

Primer sequences used for RT-PCR of common MA gene and size of PCR

Comparative Example 2-2: Result

As shown in Fig 3, the RAN efficiency of the method of the present invention is far higher than that of the conventional DTT method. The marks A and B in the Fig 3 represent MA genes detected by the DTT method and method of the present invention, respectively (* is a size marker).

Meanwhile, the results shown in the Fig 3 are represented quantitatively in the following Table 2. The method of the present invention was shown to be 16 times more sensitive than DTT method in detecting MA genes, which exist in SNU 484 cell. [Table 2]

Efficiency in detecting MA gene positive SNU484 cell

Comparative Example 3. Comparison of total RNA extraction efficiency between DTT method and method of the present invention

Total RNA extraction efficiency between the DTT method and the presently claimed method was compared by carrying out the PCR of GAPD (glyceraldehydes phosphate dehydrogenase) gene and measuring GAPD band intensity.

Comparative Example 3-1 : Experimental method

The best method for observing the RNA extraction efficiency is to measure the purity and the concentration of RNAs with a spectroscopy after extracting RNAs. However, it is impossible to prepare a sputum sample having the same number of cells because of its own property. Therefore, the present inventor(s) tried to indirectly measure the RNA extraction efficiency by extracting RNAs from a sputum sample prepared respectively using the DTT method and method of the present invention in the Comparative Example 2, followed by amplifying the GAPD genes.

(1) GAPD PCR reagent was prepared as follows. 10X PCR buffer 30

25 mM MgCl₂ 2Λβl lOO mM dNTP 30 10 pmol sense primer 10 10 pmol anti-sense primer 10 PCR enzyme 1U 10 Distilled water 22.0/^ GAPD gene was amplified under the following conditions (At this point, the reaction time should be adjusted appropriately according to a PCR apparatus, since 900 bp is generally synthesized in case of PE 2400).

94 °C, 10 minutes: 1 cycle (pre-treated at a constant temperature for Gold Taq

(PE))

94 °C, 30 seconds; 56 °C, 30 seconds; 72 °C, 1 minute: 30 cycles 72 °C, 3 minutes: 1 cycle

(3) Electrophoresis for GAPDH PCR product was carried out in 1% agarose gel containing ethidium bromide. Meanwhile, primer sequences and the sizes of PCR products used in the electrophoresis are given in the following Table 3.

[Table 3]

Primer sequences used for GAPD RT-PCR and size of PCR product

Comparative Example 3-2. Results

The result of GAPD PCR was shown in the Fig 4. More GAPD genes were shown to be amplified by the method of the present invention as compared to the DTT method. Particularly, the band obtained according to the method of the present invention was observed to be thicker as the number of SNU 484 cells increase. Such result wasn't observed in the band obtained according to the DDT method. It means that the RNA extraction efficiency of the method of the present invention is far higher than that of the DDT method. The marks A and B in the Fig 4 represent MA genes detected by the DTT method and the method of the present invention, respectively (* is a size marker).

As described in the above, the method of separating RNAs from a sputum sample according to the present invention is shown to be far more convenient, prompt and efficient as compared with the conventional method.

Claims

What is claimed is:

1. A method for extracting RNAs from a sputum sample, comprising the following steps of: (a) mixing a sputum sample with guanidinium thiocyanate buffer so that the sputum sample can be liquefied and cell-lysed; and

(b) separating RNAs from the liquefied and cell-lysed sputum sample.

2. The method according to claim 1, wherein the guanidinium thiocyanate buffer comprises 3M or more guanidinium thiocyanate.

3. The method according to claim 1, wherein the guanidinium thiocyanate buffer comprises 4M or more guanidinium thiocyanate.

4. The method according to claim 1, wherein the guanidinium thiocyanate buffer comprises 5M or more guanidinium thiocyanate.

5. The method according to claim 1, wherein the guanidinium thiocyanate buffer comprises 5M guanidinium thiocyanate, 0.1 M Tris-Cl, 1% β-mercaptoethanol, and 0.1%) dithiothreitol.

6. The method according to claim 1, wherein the step (b) comprises the following steps of; i) adding nucleic acid binding beads to the liquefied and cell-lysed sputum so that complexes of nucleic acids and beads can be formed; ii) separating the nucleic acids from the complexes; and iii) separating RNAs from the separated nucleic acids.

7. The method according to claim 6, wherein the step ii) is performed by adding a phenol-guanidinium thiocyanate reagent to the complexes of nucleic acids and beads so that the nucleic acids can be eluted.

8. The method according to claim 6, further comprising the step of eliminating non-specific bonds between impurities and nucleic acid binding beads using suitable washing buffer so that the impurities can be separated from the nucleic acid binding beads, next to step (i).

9. The method according to claim 6, wherein the step (b) comprises the following steps of:

(i) adding mRNA binding beads to the liquefied and cell-lysed sputum sample so that complexes of mRNAs and beads can be formed; and (ii) separating the mRNAs from the complexes.

10. The method according to claim 9, wherein the mRNA binding bead is a mRNA binding bead bound with biotinylated oligo (dT).

11. The method according to claim 9, wherein the mRNA binding bead is a streptavidin bead bound with biotinylated oligo (dT).

12. A kit for extracting RNAs from a sputum sample comprising guanidinium thiocyanate buffer.

13. The kit according to claim 12, wherein the guanidinium thiocyanate buffer comprises 3M or more guanidinium thiocyanate.

14. The kit according to claim 12, wherein the guanidinium thiocyanate buffer comprises 4M or more guanidinium thiocyanate.

15. The kit according to claim 12, wherein the guanidinium thiocyanate buffer comprises 5M or more guanidinium thiocyanate.

16. The kit according to claim 12, wherein the guanidinium thiocyanate buffer comprises 5M guanidinium thiocyanate, 0.1 M Tris-Cl, 1% of β-mercaptoethanol, and 0.1% dithiothreitol.

17. The kit according to claim 12 further comprising nucleic acid binding beads.

18. The kit according to claim 17, wherein the nucleic acid binding bead is mRNA binding bead.

19. The kit according to claim 18, wherein the mRNA binding bead is a mRNA binding bead bound with biotinylated oligo (dT).

20. The kit according to claim 18, wherein the mRNA binding bead is a streptavidin bead bound with biotinylated oligo (dT).

21. The kit according to claim 12 further comprising a phenol-guanidinium thiocyanate reagent.

22. The kit according to claim 12 further comprising washing buffer.