JP4599555B2 - Method for analyzing small RNA - Google Patents

Description

  The present invention relates to a method for analyzing small RNA using two-dimensional electrophoresis.

In recent years, the discovery of RNA interference (RNAi) and the fact that 22-26 nucleotide (nt) small RNAs are deeply involved in its mechanism of action have led to many small molecules. It has been elucidated that RNA or non-coding RNA (ncRNA) is present in cells and that the functions of small RNAs are very diverse. In order to grasp the whole picture of life, it is necessary to deepen understanding of small RNAs present in cells as well as genomic DNA and proteins. In order to proceed vigorously, a method capable of separating and analyzing small RNAs with excellent reproducibility is indispensable. Therefore, in the field of research, such a method is awaited. There are several methods for analyzing small RNAs, one of which is a method using two-dimensional electrophoresis. This method has already been proposed in Non-Patent Document 1. However, Non-Patent Document 1 only confirms that a mixture of several 20-nucleotide nucleotides synthesized artificially can be separated by two-dimensional electrophoresis, and exists naturally. It is not yet possible to actually isolate and analyze a mixture of small RNAs. Further, Non-Patent Document 1 does not disclose details of a specific technique for performing two-dimensional electrophoresis. In addition to the method proposed in Non-Patent Document 1, for example, there is a method proposed in Non-Patent Document 2 as an RNA analysis method using two-dimensional electrophoresis. However, when the method proposed in Non-Patent Document 2 is applied to the analysis of small RNAs, the separation of individual RNAs is poor, and in order to ensure the reproducibility of separation, skilled techniques are required. It is necessary and not easy for everyone.
Two-dimensional fractionation of complex mixture of oligonucleotides in microRNA size range: Hideaki Shiraishi, 2004 Oxford University Press, Nucleic Acids Symposium Series No. 48, 293-294 Purification of RNA Molecules by Gel techniques: Toshimichi Ikemura, Methods In Enzymology, Vol.180, pp14-25 (1989)

  Therefore, an object of the present invention is to provide an analysis method using a two-dimensional electrophoresis method capable of easily separating a low molecular weight RNA of 50 nt or less with high reproducibility.

  As a result of intensive research in view of the above points, the present inventor has obtained a gel used for performing first-dimensional electrophoresis when analyzing low-molecular RNA of 50 nt or less using two-dimensional electrophoresis. And the method for cutting out the gel of the region containing the low-molecular RNA in the electrophoresis lane, which is performed after the first-dimensional electrophoresis is performed. We have found that RNA can be easily and reproducibly separated.

The method for analyzing a low molecular weight RNA of 50 nt or less using the two-dimensional electrophoresis method of the present invention made based on the above findings, as described in claim 1, slab-like polyacrylamide having an acrylamide concentration of 12.5% to 13.5% After performing the first-dimensional electrophoresis of the sample using the gel, when cutting out the gel of the region containing the low-molecular RNA in the electrophoresis lane into a strip shape, the entire gel plate is tightly surrounded by a hydrophobic transparent film, The shape of the gel part to be cut out is approximately the same shape, and at least one side of the thin plate-like member having a hydrophobic surface is in contact with the hydrophobic transparent film located on the surface of the gel part to be cut out The gel is cut along with the hydrophobic transparent film along the peripheral edge of the thin plate member, and then the thin plate member is submerged under the gel so that the hydrophobic surface contacts the bottom surface of the cut gel, Thin plate And a member to a support removed gel, after this manner by gel polymerization for performing second dimension electrophoresis excised gel, and performing electrophoresis.
The analysis method according to claim 2 is the analysis method according to claim 1, wherein the hydrophobic surface of the thin plate member is brought into contact with the hydrophobic transparent film, and the gel and the peripheral portion of the thin plate member together with the hydrophobic transparent film. It is characterized by cutting along.
Further, the analysis method according to claim 3, in the analysis method according to claim 1 or 2, by comparing the migration patterns of samples prepared using individuals or cells under at least two specific conditions, The expression characteristics of the small RNA are comparatively analyzed.
Further, a kit for performing the analysis method according to any one of claims 1 to 3 of the present invention, as described in claim 4, a gel plate and a gel-forming reagent for performing first-dimensional electrophoresis, Gel plate and gel forming reagent for performing second-dimensional electrophoresis, comb for forming sample injection groove in gel when performing first-dimensional electrophoresis, hydrophobic transparent film, on at least one surface It comprises at least a thin plate-like member having a hydrophobic surface.

  According to the present invention, there is provided an analysis method using a two-dimensional electrophoresis method capable of easily separating a low molecular weight RNA of 50 nt or less with high reproducibility.

  In the method of analyzing low molecular weight RNA of 50 nt or less using the two-dimensional electrophoresis method of the present invention, the first-dimensional electrophoresis of a sample was performed using a slab-like polyacrylamide gel having an acrylamide concentration of 12.5% to 13.5%. Thereafter, when the gel of the region containing the low-molecular RNA in the electrophoresis lane is cut out in a strip shape, the entire gel plate is tightly surrounded by a hydrophobic transparent film, and is substantially the same shape as the shape of the gel portion to be cut out, at least Either surface of the thin plate-like member having a hydrophobic surface on one side is brought into contact with the hydrophobic transparent film located on the surface of the gel part to be cut out, and the gel and the peripheral portion of the thin plate-like member together with the hydrophobic transparent film Then, the thin plate member is submerged under the gel so that the hydrophobic surface is in contact with the bottom surface of the cut gel, and the gel is taken out using the thin plate member as a support. After the gel was allowed to gel polymerization for performing second dimensional electrophoresis cut Te, and performing electrophoresis.

  In the present invention, the gel used for the first-dimensional electrophoresis of the sample is specified as a slab-like polyacrylamide gel having an acrylamide concentration of 12.5% to 13.5%. The reason why the type of gel is identified as polyacrylamide gel is that it is excellent in the separation ability for a mixture of small RNAs of 50 nt or less to be analyzed. The acrylamide concentration is specified as 12.5% to 13.5% because when the concentration falls below 12.5%, it becomes a condition that improves resolution for a mixture of RNA larger than 50 nt, and conversely, it is as low as 50 nt or less. This is because the separation ability for a mixture of molecular RNAs decreases. On the other hand, when the concentration exceeds 13.5%, when performing second-dimensional electrophoresis under the conditions described below, the difference from the acrylamide concentration of the gel used for the second-dimensional electrophoresis is reduced. This is because the resolution of RNA spots obtained after eye electrophoresis is deteriorated, making it difficult to identify individual spots.

  In the present invention, after performing the first-dimensional electrophoresis of the sample, when the gel of the region containing the low-molecular RNA in the electrophoresis lane is cut out in a strip shape, the entire gel plate is closely enclosed with a hydrophobic transparent film, The shape of the gel part to be cut out is approximately the same shape, and at least one side of the thin plate-like member having a hydrophobic surface is in contact with the hydrophobic transparent film located on the surface of the gel part to be cut out The gel is cut along with the hydrophobic transparent film along the peripheral edge of the thin plate member, and then the thin plate member is submerged under the gel so that the hydrophobic surface contacts the bottom surface of the cut gel, The gel is taken out using the thin plate member as a support. When the gel is cut into strips, the cut-out length is preferably shorter than the lateral width of the gel used for the second-dimensional electrophoresis, and the cut-out width is preferably narrower than the width of the electrophoresis lane. By carrying out as described above, for example, even when an extremely fine strip-like gel having a cutting length of 10 to 20 cm and a cutting width of 0.3 to 0.7 cm is cut out, it can be cut out easily and accurately. .

  Examples of the hydrophobic transparent film used for tightly enclosing the entire gel plate include a food wrapping transparent wrap film made of polyvinylidene chloride. If a hydrophilic transparent film such as cellophane is used instead of the hydrophobic transparent film, the gel plate softens and expands with moisture from the electrophoresis buffer remaining on the surface of the gel. It is difficult to tightly enclose the gel, and as a result, the gel cannot be cut out with high accuracy.

  The thin plate-like member having a hydrophobic surface on at least one surface may be any gel plate as long as it has a hydrophobic surface required when taking out the cut gel, which is performed later. It is preferable that the surface of the thin plate member to be brought into contact with the hydrophobic transparent film used for tightly enclosing the whole has affinity with the hydrophobic transparent film (the transparent transparent film for food packaging made of polyvinylidene chloride as the hydrophobic transparent film) When a wrap film is used, the hydrophobic surface of the thin plate member has an affinity with the hydrophobic transparent film). When the gel is cut along with the hydrophobic transparent film along the peripheral edge of the thin plate member, slipping between the hydrophobic transparent film and the thin plate member is suppressed, and a gel having a straight cut is simply and accurately cut out. This is because it becomes possible. In addition, the thin plate-like member is made to be a support by allowing the thin plate-like member to be submerged under the gel so that its hydrophobic surface is in contact with the bottom surface of the cut gel, and taking the gel with the thin plate-like member as a support. Thus, the cut gel can be easily moved without breaking. If the thin plate-like member has a hydrophilic surface, and the same operation is performed using this hydrophilic surface and the gel is taken out, the moisture present on the gel surface interferes with the thin plate-like member. Since it is difficult to accurately sink under the cut gel, it is difficult to set the gel on the gel plate used for the second-dimensional electrophoresis.

  The gel used for the second-dimensional electrophoresis is preferably a slab-like polyacrylamide gel having an acrylamide concentration of 21.0% to 23.0% and a urea concentration of 6.0M to 8.0M, for example. The reason why the type of gel is polyacrylamide gel is that it is excellent in the separation ability for a mixture of small RNAs of 50 nt or less to be analyzed. It is preferable that the acrylamide concentration is 21.0% to 23.0%. If the concentration is lower than 21.0%, the difference from the acrylamide concentration of the gel used for the first dimensional electrophoresis is reduced. This is because the resolution of RNA spots obtained after electrophoresis is deteriorated, making it difficult to grasp individual spots. On the other hand, if the concentration exceeds 23.0%, the separability of RNA in the vicinity of 50 nt deteriorates, and the gel becomes fragile, which makes it difficult to handle. The reason why the urea concentration is preferably 6.0 M to 8.0 M is that the influence on the second-dimensional electrophoresis due to the difference in the secondary structure of the small RNA can be eliminated. By adding urea with a concentration of 6.0M to 8.0M to the gel used for the second dimension electrophoresis without adding urea to the gel used for the first dimension electrophoresis, small RNAs of the same size can be obtained. It can be separated by the difference in secondary structure.

  In addition, the preparation method of the sample and gel containing low molecular RNA of 50 nt or less, and the operation method of electrophoresis may be carried out by a method known per se (if necessary, “Polyacrylamide Gel Electrophoresis” in Molecular Cloning, a laboratory manual, second edition: See J. Sambrook, EF Fitsch and T. Maniatis (1989), pp. 6.36-6.48, Cold Spring Harbor Laboratory Press, New York.).

  According to the present invention, a low molecular weight RNA of 50 nt or less (approximately 15 nt to 50 nt) can be easily and reproducibly separated on a second-dimensional gel. Thus, for at least some 50 nt or smaller small RNAs, they can be obtained with high purity as each spot on the gel, thus facilitating their sequencing and thus new 50 nt or less Efficient search for small RNA is possible. In addition, two-dimensional electrophoresis of samples prepared using individuals or cells under at least two specific conditions can be performed, and the analysis characteristics of small RNAs can be compared and analyzed by comparing the migration patterns. As a result, it becomes easy to clarify what kind of small RNAs are involved in higher life phenomena such as aging, behavior, neurogenesis and embryogenesis in multicellular organisms.

  The method for analyzing low molecular weight RNA of 50 nt or less using the two-dimensional electrophoresis method of the present invention includes a gel plate and a gel-forming reagent for performing the first-dimensional electrophoresis, and a method for performing the second-dimensional electrophoresis. At least a gel plate and a gel-forming reagent, a comb for forming a sample injection groove in the gel when performing first-dimensional electrophoresis, a hydrophobic transparent film, and a thin plate-like member having a hydrophobic surface on at least one surface By using the kit comprising it, anyone can carry out easily and with good reproducibility. Here, the gel plate for performing the first-dimensional electrophoresis and the gel plate for performing the second-dimensional electrophoresis are, for example, made of glass or resin approximately 50 cm long × 20 cm wide. Examples of the gel forming reagent for performing the first-dimensional electrophoresis include an acrylamide solution prepared by mixing the following components and preparing a total amount of 213 ml with sterile water.

Acrylamide 26.6g
N, N'-methylenebisacrylamide 1.4g
Tris base 0.65g
Boric acid 0.33g
0.5M EDTA (pH8.0) 0.24ml
Ammonium persulfate 0.2g
N, N, N ', N'-Tetramethylethylenediamine (TEMED) 230μl

  Examples of the gel forming reagent for performing the second-dimensional electrophoresis include an acrylamide solution prepared by mixing the following components and preparing a total amount of 240 ml with sterilized water.

Acrylamide 50.2g
N, N'-methylenebisacrylamide 2.6g
Urea 100g
Tris base 0.78g
Boric acid 0.40g
0.5M EDTA (pH8.0) 0.29ml
Ammonium persulfate 0.1g
N, N, N ', N'-Tetramethylethylenediamine (TEMED) 230μl

  As a comb for forming a sample injection groove in the gel during the first dimensional electrophoresis, for example, there are 10 teeth of 1 cm in length × 1 cm in width at intervals of 0.2 cm on one side and teeth on the other side. However, there is a Teflon (registered trademark) plate having a thickness of 0.2 cm. Examples of the hydrophobic transparent film include a transparent wrap film for food packaging made of polyvinylidene chloride. Examples of the thin plate-like member having a hydrophobic surface on at least one surface include, for example, a plastic plate that is hydrophobic on both surfaces (both surfaces are subjected to hydrophobic treatment), and one surface that is used in the examples described later is hydrophobic. One example is Gel Bond PAG film manufactured by Pharmacia Biotech, which has a hydrophilic surface on the other side.

Hereinafter, the present invention will be described in detail by way of examples using nematodes ( C. elegans ), but the present invention is not construed as being limited to the following description.

Experiment 1:
(Preparation of RNA sample for electrophoresis)
10 g of nematode was treated with 10 ml of TRIzol reagent (Invitrogen) to obtain 0.5 mg of crude RNA extract. This was fractionated by a stepwise 2-propanol precipitation method to obtain 5 μg of a fraction containing RNA of 100 nt or less. In order to label the 3 'end of RNA with a radioisotope, 2.5 µg of this RNA fraction, 6.5 µl of ³² P-pCp (111 TBq / mmol), 20 unit of T4 RNA ligase (Takara Shuzo), final concentration 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM DTT, and 1 mM ATP were added to 15 μl with sterilized water and reacted at 4 ° C. for 16 to 20 hours. For electrophoresis, add 5 μl of glycerol buffer for electrophoresis (containing 50% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol) and 5 μl of sterilized water to 15 μl of RNA fraction labeled at the 3 ′ end in this way. An RNA sample was used.

(Two-dimensional electrophoresis)
In order to perform the first-dimensional electrophoresis, a slab-like polyacrylamide gel having an acrylamide concentration of 13.1% was prepared as follows. Specifically, 70 ml of 40% acrylamide solution (a solution obtained by dissolving 380 g of acrylamide and 20 g of N, N′-methylenebisacrylamide in water to 1000 ml), 10 × TBE (108 g of Tris base, 55 g of boric acid, 40 ml of 0.5M EDTA, pH 8. 0) Add 6 ml and 135 ml of water to a 300 ml beaker and mix with a stirrer to the extent that it does not foam. Add TEMED 230 μl and 2 ml of 10% ammonium persulfate to this glass and mix quickly. 47.5 cm x 19.5 cm wide x 0.2 cm gap). After the acrylamide polymerization reaction was completed and the gel was solidified, the gel plate was set in an electrophoresis apparatus, and pre-energization was performed for 1 hour at 1000 V (constant voltage). The electrophoresis was performed using 0.3 × TBE as the electrophoresis buffer and a heat sink attached to the gel plate. After pre-energization, place the electrophoresis RNA sample in the gel sample injection groove (width 1 cm x depth 1 cm x thickness 0.2 cm), and at room temperature until the bromophenol blue contained in the sample flows out from the gel at a constant current of 10 mA. (See FIG. 1 (1): In the figure, 1D indicates the first-dimensional electrophoresis direction. The same applies hereinafter).

  After the first-dimensional electrophoresis was completed, the gel plate was removed from the electrophoresis apparatus, and the entire gel plate was tightly enclosed with a transparent wrap film for food packaging made of polyvinylidene chloride, which is a hydrophobic transparent film. Autoradiography was performed after the fluorescent tape was marked on the wrap film (using Fuji Photo Film's trade name: FUJI X-ray film RA). The exposure was performed at -80 ° C. for 16 to 24 hours using an intensifying screen. Align the fluorescent tape mark on the wrap film with the fluorescent tape position marked on the photosensitized autoradiogram, and the gel part of the region containing small RNA of 50 nt or less that was photodetected (the width of the electrophoresis lane is 1 cm) Product name manufactured by Pharmacia Biotech, which is a thin plate member cut into a length of 15.5 cm and a width of 0.5 cm, with one surface being a hydrophobic surface and the other surface being a hydrophilic surface. The hydrophobic surface of the Gel Bond PAG film was brought into contact, and the gel was cut along with the wrap film along the periphery of the thin plate member using a cutter knife. Next, the thin plate member was submerged under the gel so that the hydrophobic surface was in contact with the bottom surface of the cut gel, and the gel was taken out using the thin plate member as a support (see FIG. 1 (2)). . Note that the gel cut-out width was made narrower than the width of the electrophoresis lane because the low-molecular weight of 50 nt or less present in the first-dimensional gel cut into a strip shape immediately after the second-dimensional electrophoresis performed later. This is because the low-molecular RNA is finally separated on the second-dimensional gel with high reproducibility by moving the RNA to the second-dimensional gel.

  Two hours before the completion of the first-dimensional electrophoresis, preparation for the second-dimensional electrophoresis was performed. Because the gel is dry and fragile, immediately after the first-dimensional gel is cut out, it is ready to be polymerized with the gel for second-dimensional electrophoresis. This is because it is preferable. A slab-like polyacrylamide gel having an acrylamide concentration of 22.0% and a urea concentration of 7.0 M was used as the gel for performing the second-dimensional electrophoresis. This gel was prepared with a composition of 132 ml of 40% acrylamide solution, 7.2 ml of 10 × TBE, 7 ml of water and 100 g of urea. Each component was placed in a 300 ml beaker and urea was completely dissolved over about 1 hour. After the urea is completely dissolved, the resulting solution is filtered and degassed, poured into a 300 ml beaker and mixed with a stirrer to the extent that no foaming occurs, where N, N, N ′, N′-tetramethylethylenediamine ( TEMED) After adding 230 μl and 1% 10% ammonium persulfate and mixing quickly, the resulting solution was subjected to second-dimensional electrophoresis, in which a first-dimensional gel cut into strips using a thin plate member was set. The glass gel plate used (47.5 cm long x 19.5 cm wide x 0.2 cm gap) was prepared by gently pouring the polymer so that no bubbles would enter under the first-dimensional gel.

  After the acrylamide polymerization reaction is completed and the gel hardens, place the gel plate in the electrophoresis apparatus (at least at this stage, the wrap film cut with the gel remaining on the surface of the first dimension gel ), The buffer tanks at the top and bottom of the gel plate are filled with 0.3 × TBE, and the electrophoresis urea buffer (7M urea, 0.25% bromophenol blue, 0.25% xylene cyanol) is used at the top of the gel. Was mounted for about 24 hours under conditions of 10 mA constant current and room temperature until xylene cyanol contained in the electrophoresis urea buffer moved 30 cm below the first dimension gel (Fig. 1 (3 ) Reference: In the figure, 2D indicates the direction of electrophoresis in the second dimension (the same applies hereinafter). After the completion of the second-dimensional electrophoresis, autoradiography was performed in the same manner as when the first-dimensional electrophoresis was completed. As a result, it was possible to separate many small RNAs on the second-dimensional gel (see FIG. 1 (4)).

Experiment 2:
A sample prepared from an embryonic stage nematode (a nematode before hatching) and a mixed stage nematode (a nematode in one of the larval stages L1 to L4 from egg to adult) are mixed. Samples prepared from the above were subjected to two-dimensional electrophoresis in the same manner as in Experiment 1, and low-molecular RNAs of 50 nt or less were separated on the second-dimensional gel. The results are shown in FIG. 2 (1) and (2), respectively (in the figure, 1D indicates the first-dimensional electrophoresis direction and 2D indicates the second-dimensional electrophoresis direction). When both were compared, it was found that the electrophoretic images were different from each other, and the expression state of small RNA in vivo was different at each stage. There are 85 spots that are specific to the embryonic stage (for example, those shown by arrows in Fig. 2 (1)), and spots that are specific to the mixed stage (for example, those shown by arrows in Fig. 2 (2)). There were 51 and 54 that were commonly seen (for example, those circled). From the spots thus obtained, it was possible to search for a novel low molecular weight RNA of 50 nt or less.

  The present invention has industrial applicability in that it can provide an analysis method using a two-dimensional electrophoresis method that can easily separate a low molecular weight RNA of 50 nt or less with good reproducibility. .

It is a figure which shows the schematic procedure of the analysis method of 50 nt or less small molecule RNA using the two-dimensional electrophoresis method of this invention in an Example. The figure shows two-dimensional electrophoresis images of small RNAs of the nematode in the embryonic stage and two-dimensional electrophoresis images of small RNAs of the mixed stage nematode.

Claims (4)

  This is a method for analyzing small RNAs of 50 nucleotides or less using two-dimensional electrophoresis, and the first-dimensional electrophoresis of the sample was performed using a slab-like polyacrylamide gel with an acrylamide concentration of 12.5% to 13.5% Thereafter, when the gel of the region containing the low-molecular RNA in the electrophoresis lane is cut out in a strip shape, the entire gel plate is tightly surrounded by a hydrophobic transparent film, and is substantially the same shape as the shape of the gel portion to be cut out, at least Either surface of the thin plate-like member having a hydrophobic surface on one side is brought into contact with the hydrophobic transparent film located on the surface of the gel part to be cut out, and the gel and the peripheral portion of the thin plate-like member together with the hydrophobic transparent film Then, the thin plate member is submerged under the gel so that the hydrophobic surface contacts the bottom surface of the cut gel, and the gel is taken out using the thin plate member as a support. After gel and polymerized for performing second dimension electrophoresis excised gel as analytical methods and performing electrophoresis.   2. The analysis method according to claim 1, wherein the hydrophobic surface of the thin plate member is brought into contact with the hydrophobic transparent film, and the gel is cut along the peripheral edge of the thin plate member together with the hydrophobic transparent film.   3. The expression characteristic of the small RNA is comparatively analyzed by comparing the migration patterns of samples prepared using individuals or cells under at least two specific conditions. Analysis method.   A kit for performing the analysis method according to any one of claims 1 to 3, comprising a gel plate and a gel-forming reagent for performing first-dimensional electrophoresis, and a second-dimensional electrophoresis. At least a gel plate and a gel-forming reagent, a comb for forming a sample injection groove in the gel when performing first-dimensional electrophoresis, a hydrophobic transparent film, and a thin plate-like member having a hydrophobic surface on at least one surface A kit characterized by comprising.