JP2006189359A - Analytical method for low-molecular rna - Google Patents

Analytical method for low-molecular rna Download PDF

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JP2006189359A
JP2006189359A JP2005002336A JP2005002336A JP2006189359A JP 2006189359 A JP2006189359 A JP 2006189359A JP 2005002336 A JP2005002336 A JP 2005002336A JP 2005002336 A JP2005002336 A JP 2005002336A JP 2006189359 A JP2006189359 A JP 2006189359A
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gel
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Chisato Ushida
千里 牛田
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Hirosaki University NUC
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Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical method using a two-dimensional electrophoresis capable of separating easily 50nt or less of low-molecular RNA with excellent reproducibility. <P>SOLUTION: When cutting down strip-likely gel of an area including the low-molecular RNA in a migration lane, after carrying out one-dimensional electrophoresis for a sample, using a slab-like polyacrylamide gel having 12.5%-13.5% of acrylamide concentration, the whole gel sheet is enveloped closely with a hydrophobic transparent film, either of faces of a thin-sheet-like member having a shape substantially same to that of the cut-down gel portion, and having a hydrophobic face at least one side face, is brought into contact with the hydrophobic transparent film positioned on the surface of the cut-down gel portion to cut the gel along a peripheral edge part of the thin-sheet-like member together with the hydrophobic transparent film, then the thin-sheet-like member is hidden under the gel to bring the hydrophobic face into contact with a bottom face of the cut-in gel, the gel is taken out using the thin-sheet-like member as a support body, the gel cut down by this manner is layered with a gel for two-dimensional electrophoresis, and the electrophoresis is carried out thereafter. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は、二次元電気泳動法を用いた低分子RNAの分析方法に関する。   The present invention relates to a method for analyzing small RNA using two-dimensional electrophoresis.

近年、RNA干渉（RNA interference, RNAi）の発見と、その作用機構に22〜26ヌクレオチド（nt）の低分子RNAが深く関与していることが明らかにされたことをきっかけに、数多くの低分子RNAあるいは非翻訳RNA（non-coding RNA, ncRNA）が細胞に存在すること、低分子RNAの機能は非常に多岐にわたることが解明されつつある。生命の全体像を把握するためには、ゲノムDNAや蛋白質と同様に、細胞に存在する低分子RNAについても理解を深めることが必要であるところ、このような状況下において、低分子RNAの研究を精力的に進めていくためには、低分子RNAを優れた再現性のもとに分離して分析することができる方法が不可欠となる。従って、研究の現場では、このような方法が待ち望まれている。低分子RNAの分析方法としては、いくつかの方法が考えられるが、その1つに二次元電気泳動法を用いた方法がある。この方法については既に非特許文献1において提案がされている。しかしながら、非特許文献1においては、人工的に合成したいくつかの20数ヌクレオチドの低分子RNAの混合物が、二次元電気泳動法により分離されることを確認しているに過ぎず、天然に存在する低分子RNAの混合物を実際に分離して分析するまでには至っていない。また、非特許文献1においては、二次元電気泳動法を行うための具体的な手法の詳細については明らかにされていない。二次元電気泳動法を用いたRNAの分析方法としては、非特許文献1において提案されている方法以外にも、例えば、非特許文献2において提案されている方法がある。しかしながら、非特許文献2において提案されている方法は、低分子RNAの分析に適用した場合、個々のRNA同士の分離が悪く、また、分離の再現性を確保するためには、熟練した技術が必要であり、誰もが簡便に行うことができるものではない。
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) 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 is being 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 the understanding of small RNAs present in cells as well as genomic DNA and proteins. In order to proceed vigorously, a method that can separate and analyze 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 confirmed 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, in Non-Patent Document 1, details of a specific method for performing two-dimensional electrophoresis are not disclosed. 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)

そこで本発明は、50nt以下の低分子RNAを簡便に再現性よく分離することができる二次元電気泳動法を用いた、その分析方法を提供することを目的とする。   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.

本発明者は、上記の点に鑑みて鋭意研究を重ねた結果、二次元電気泳動法を用いて50nt以下の低分子RNAの分析を行う際、一次元目の電気泳動を行うために用いるゲルを特定し、かつ、一次元目の電気泳動を行った後に行う、泳動レーン中の前記低分子RNAを含む領域のゲルの切り出し方法を特定することで、二次元目のゲル上で前記低分子RNAを簡便に再現性よく分離することができることを見い出した。   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.

上記の知見に基づいてなされた本発明の二次元電気泳動法を用いた50nt以下の低分子RNAの分析方法は、請求項1記載の通り、アクリルアミド濃度が12.5%〜13.5%のスラブ状ポリアクリルアミドゲルを用いて試料の一次元目の電気泳動を行った後、泳動レーン中の前記低分子RNAを含む領域のゲルをストリップ状に切り出す際、ゲル板全体を疎水性透明フィルムで密着包囲し、切り出すゲルの部分の形状と略同一形状であって、少なくとも一方の面に疎水性面を有する薄板状部材のいずれかの面を、切り出すゲルの部分の表面に位置する疎水性透明フィルムに当接させ、ゲルを疎水性透明フィルムとともに薄板状部材の周縁部に沿って切り込み、次に、薄板状部材を、切り込んだゲルの底面にその疎水性面が当接するようにゲルの下に潜り込ませ、薄板状部材を支持体にしてゲルを取り出し、このようにして切り出したゲルを二次元目の電気泳動を行うためのゲルと重合させた後、電気泳動を行うことを特徴とする。
また、請求項2記載の分析方法は、請求項1記載の分析方法において、薄板状部材の疎水性面を疎水性透明フィルムに当接させ、ゲルを疎水性透明フィルムとともに薄板状部材の周縁部に沿って切り込むことを特徴とする。
また、請求項3記載の分析方法は、請求項1または2記載の分析方法において、少なくとも2種類の特定の条件下にある個体または細胞を用いて調製した試料の泳動パターンを比較することで、前記低分子RNAの発現特性を比較分析することを特徴とする。
また、本発明の請求項1乃至3のいずれかに記載の分析方法を行うためのキットは、請求項4記載の通り、一次元目の電気泳動を行うためのゲル板とゲル形成用試薬、二次元目の電気泳動を行うためのゲル板とゲル形成用試薬、一次元目の電気泳動を行う際にゲルに試料注入溝を形成するための櫛、疎水性透明フィルム、少なくとも一方の面に疎水性面を有する薄板状部材を少なくとも含んでなることを特徴とする。 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 Cutting the gel along with the hydrophobic transparent film along the periphery of the thin plate member, and then letting the thin plate member sink under the gel so that the hydrophobic surface abuts 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.

本発明によれば、50nt以下の低分子RNAを簡便に再現性よく分離することができる二次元電気泳動法を用いた、その分析方法が提供される。   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.

本発明の二次元電気泳動法を用いた50nt以下の低分子RNAの分析方法は、アクリルアミド濃度が12.5%〜13.5%のスラブ状ポリアクリルアミドゲルを用いて試料の一次元目の電気泳動を行った後、泳動レーン中の前記低分子RNAを含む領域のゲルをストリップ状に切り出す際、ゲル板全体を疎水性透明フィルムで密着包囲し、切り出すゲルの部分の形状と略同一形状であって、少なくとも一方の面に疎水性面を有する薄板状部材のいずれかの面を、切り出すゲルの部分の表面に位置する疎水性透明フィルムに当接させ、ゲルを疎水性透明フィルムとともに薄板状部材の周縁部に沿って切り込み、次に、薄板状部材を、切り込んだゲルの底面にその疎水性面が当接するようにゲルの下に潜り込ませ、薄板状部材を支持体にしてゲルを取り出し、このようにして切り出したゲルを二次元目の電気泳動を行うためのゲルと重合させた後、電気泳動を行うことを特徴とする。   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.

本発明では、試料の一次元目の電気泳動を行うために用いるゲルをアクリルアミド濃度が12.5%〜13.5%のスラブ状ポリアクリルアミドゲルと特定する。ゲルの種類をポリアクリルアミドゲルと特定するのは、分析対象の50nt以下の低分子RNAの混合物に対する分離能に優れるからである。アクリルアミド濃度を12.5%〜13.5%と特定するのは、濃度が12.5%を下回ると、それは50ntよりも大きいRNAの混合物に対する分離能を向上させるような条件になってしまい、反対に50nt以下の低分子RNAの混合物に対する分離能が低下するからである。一方、濃度が13.5%を上回ると、後述するような条件で二次元目の電気泳動を行う場合、二次元目の電気泳動に用いるゲルのアクリルアミド濃度との差が縮まってしまうことで、二次元目の電気泳動を行った後に得られるRNAスポットの分離能が悪くなり、個々のスポットの把握が困難になるからである。   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.

本発明では、試料の一次元目の電気泳動を行った後、泳動レーン中の前記低分子RNAを含む領域のゲルをストリップ状に切り出す際、ゲル板全体を疎水性透明フィルムで密着包囲し、切り出すゲルの部分の形状と略同一形状であって、少なくとも一方の面に疎水性面を有する薄板状部材のいずれかの面を、切り出すゲルの部分の表面に位置する疎水性透明フィルムに当接させ、ゲルを疎水性透明フィルムとともに薄板状部材の周縁部に沿って切り込み、次に、薄板状部材を、切り込んだゲルの底面にその疎水性面が当接するようにゲルの下に潜り込ませ、薄板状部材を支持体にしてゲルを取り出す。ゲルをストリップ状に切り出す際、その切り出し長さは二次元目の電気泳動に用いるゲルの横幅より短くするのが好ましく、切り出し幅は泳動レーンの幅より狭くするのが好ましいが、ゲルの切り出しを上記のようにして行うことで、例えば、切り出し長さが10〜20cmで切り出し幅が0.3〜0.7cmといったような極細のストリップ状のゲルを切り出す場合でも、簡便に精度よく切り出すことが可能となる。   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. .

ゲル板全体を密着包囲するために用いる疎水性透明フィルムとしては、例えば、ポリ塩化ビニリデン製の食品包装用透明ラップフィルムが挙げられる。仮に、疎水性透明フィルムに代わりにセロハンのような親水性透明フィルムを用いた場合、当該フィルムがゲルの表面に残存する電気泳動用バッファー由来の水分を含んで軟化膨張したりしてゲル板全体を密着包囲することが困難となり、その結果、ゲルの切り出しを精度よく行えないといった問題を招来する。   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.

少なくとも一方の面に疎水性面を有する薄板状部材は、後に行う、切り込んだゲルを取り出す際に必要となる疎水性面を有するものであればどのようなものであってもよいが、ゲル板全体を密着包囲するために用いる疎水性透明フィルムに当接させる薄板状部材の面は、疎水性透明フィルムと親和性を有することが好ましい（疎水性透明フィルムとしてポリ塩化ビニリデン製の食品包装用透明ラップフィルムを用いる場合、薄板状部材の疎水性面は、疎水性透明フィルムと親和性を有する）。ゲルを疎水性透明フィルムとともに薄板状部材の周縁部に沿って切り込む際、疎水性透明フィルムと薄板状部材の間での滑りが抑えられ、直線状の切口を有するゲルを簡便に精度よく切り出すことが可能となるからである。また、薄板状部材を、切り込んだゲルの底面にその疎水性面が当接するようにゲルの下に潜り込ませ、薄板状部材を支持体にしてゲルを取り出すことで、薄板状部材を支持体にして、切り込んだゲルを崩すことなく容易に移動させることができる。仮に、薄板状部材が親水性面を有し、この親水性面を利用して同様の操作を行ってゲルを取り出そうとした場合、ゲル表面に存在する水分が邪魔をして、薄板状部材を切り込んだゲルの下に正確に潜り込ませることが困難になることで、ゲルを二次元目の電気泳動に用いるゲル板にセットすることが困難となるといった問題を招来する。   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.

二次元目の電気泳動を行うために用いるゲルは、例えば、アクリルアミド濃度が21.0%〜23.0%で尿素濃度が6.0M〜8.0Mのスラブ状ポリアクリルアミドゲルが好ましい。ゲルの種類をポリアクリルアミドゲルとすることが好ましいのは、分析対象の50nt以下の低分子RNAの混合物に対する分離能に優れるからである。アクリルアミド濃度を21.0%〜23.0%とすることが好ましいのは、濃度が21.0%を下回ると、一次元目の電気泳動に用いるゲルのアクリルアミド濃度との差が縮まってしまうことで、二次元目の電気泳動を行った後に得られるRNAスポットの分離能が悪くなり、個々のスポットの把握が困難になるからである。一方、濃度が23.0%を上回ると、50nt付近のRNAの分離能が悪くなったり、ゲルが壊れやすくなることで、その取り扱いが困難になったりするからである。尿素濃度を6.0M〜8.0Mとすることが好ましいのは、低分子RNAの二次構造の違いによる二次元目の電気泳動への影響をなくすことができるからである。一次元目の電気泳動に用いるゲルに尿素を添加せず、二次元目の電気泳動に用いるゲルに濃度が6.0M〜8.0Mの尿素を添加することで、同じ大きさの異なる低分子RNAを二次構造の違いにより分離することができる。   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.

なお、50nt以下の低分子RNAを含む試料やゲルの調製方法、電気泳動の操作方法は自体公知の方法で行えばよい（必要であれば“Polyacrylamide Gel Electrophoresis” in Molecular Cloning, a laboratory manual, second edition: J. Sambrook, E. F. Fitsch and T. Maniatis (1989) , pp. 6.36-6.48, Cold Spring Harbor Laboratory Press, New York.を参照のこと）。   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.).

本発明によれば、二次元目のゲル上で、50nt以下（おおよそ15nt〜50nt）の低分子RNAを簡便に再現性よく分離することができる。従って、少なくともいくつかの50nt以下の低分子RNAについては、それらをゲル上の各スポットとして高い精製度の下に取得することができることから、その配列決定を容易にし、よって、新規な50nt以下の低分子RNAの効率的な探索が可能となる。また、少なくとも2種類の特定の条件下にある個体または細胞を用いて調製した試料の二次元電気泳動を行い、その泳動パターンを比較することで、低分子RNAの発現特性を比較分析することができることから、多細胞生物における老化、行動、神経発生、胚発生などの高次生命現象にどのような低分子RNAがどのように関わっているのかについての解明が容易となる。   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.

本発明の二次元電気泳動法を用いた50nt以下の低分子RNAの分析方法は、一次元目の電気泳動を行うためのゲル板とゲル形成用試薬、二次元目の電気泳動を行うためのゲル板とゲル形成用試薬、一次元目の電気泳動を行う際にゲルに試料注入溝を形成するための櫛、疎水性透明フィルム、少なくとも一方の面に疎水性面を有する薄板状部材を少なくとも含んでなるキットを用いることで、誰もが簡便に再現性よく行うことができる。ここで、一次元目の電気泳動を行うためのゲル板と、二次元目の電気泳動を行うためのゲル板は、例えば、縦50cm×横20cm程度のガラス製または樹脂製である。一次元目の電気泳動を行うためのゲル形成用試薬としては、例えば、下記の成分を混合して滅菌水で総量213mlに調製したアクリルアミド溶液が挙げられる。   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.

アクリルアミド 26.6g
N,N’-メチレンビスアクリルアミド 1.4g
トリス塩基 0.65g
ホウ酸 0.33g
0.5M EDTA（pH8.0） 0.24ml
過硫酸アンモニウム 0.2g
N,N,N',N'-テトラメチルエチレンジアミン（TEMED） 230ml 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) 230ml

また、二次元目の電気泳動を行うためのゲル形成用試薬としては、例えば、下記の成分を混合して滅菌水で総量240mlに調製したアクリルアミド溶液が挙げられる。   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.

アクリルアミド 50.2g
N,N’-メチレンビスアクリルアミド 2.6g
尿素 100g
トリス塩基 0.78g
ホウ酸 0.40g
0.5M EDTA（pH8.0） 0.29ml
過硫酸アンモニウム 0.1g
N,N,N',N'-テトラメチルエチレンジアミン（TEMED） 230ml 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) 230ml

一次元目の電気泳動を行う際にゲルに試料注入溝を形成するための櫛としては、例えば、一方に縦1cm×横1cmの歯を0.2cm間隔で10本もち、他方には歯をもたない、厚さ0.2cmのテフロン（登録商標）製の板が挙げられる。疎水性透明フィルムとしては、例えば、ポリ塩化ビニリデン製の食品包装用透明ラップフィルムが挙げられる。少なくとも一方の面に疎水性面を有する薄板状部材としては、例えば、両面が疎水性である（両面が疎水処理された）プラスチック板や、後述する実施例で用いた、一方の面が疎水性面で他方の面が親水性面であるPharmacia Biotech社製の商品名：Gel Bond PAG filmが挙げられる。   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.

以下、本発明を線虫（C. elegans）を用いた実施例によって詳細に説明するが、本発明は、以下の記載に何ら限定して解釈されるものではない。 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.

実験1：
（泳動用RNA試料の準備）
線虫10gをTRIzol試薬（Invitrogen社製）10mlで処理し、粗RNA抽出物0.5mgを得た。これをステップワイズの2-プロパノール沈殿法により分画し、100nt以下のRNAを含む画分5μgを得た。RNAの3'末端を放射性同位体で標識するために、このRNA画分2.5μgに、6.5μlの32P-pCp（111TBq/mmol）、20unitのT4 RNA ligase （宝酒造社製）、終濃度50mMのTris-HCl（pH7.5）、10mMのMgCl2、10mMのDTT、1mMのATPを加え、滅菌水で15μlにあわせて、4℃で16時間〜20時間反応させた。このようにして3'末端を標識したRNA画分15μlに、電気泳動用グリセロールバッファー（50%グリセロールと0.25%ブロモフェノールブルーと0.25%キシレンシアノール含有）を5μl、滅菌水を5μl加えて泳動用RNA試料とした。 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.

（二次元電気泳動）
一次元目の電気泳動を行うために、以下のようにしてアクリルアミド濃度が13.1%のスラブ状ポリアクリルアミドゲルを作製した。即ち、40%アクリルアミド溶液（アクリルアミド380gとN,N'-メチレンビスアクリルアミド20gを水に溶かして1000mlにした溶液）70ml、10×TBE（トリス塩基108g，ホウ酸55g，0.5M EDTA 40ml，pH8.0）6ml、水135mlを300mlビーカーに加えて泡立てない程度にスターラーで混ぜ、ここにTEMED230μlと10%過硫酸アンモニウム2mlを加えて素早く混ぜた後、得られた溶液をセットアップしたガラス製ゲル板（縦47.5cm×横19.5cm×隙間0.2cm）に流し込んだ。アクリルアミドの重合反応が終了してゲルが固まった後、ゲル板を泳動装置にセットして、1000V（定電圧）、1時間の予備通電を行った。泳動バッファーには0.3×TBEを用い、ゲル板には放熱板を付けて泳動を行った。予備通電終了後、泳動用RNA試料をゲルの試料注入溝（幅1cm×深さ1cm×厚さ0.2cm）に入れ、10mA定電流で、試料に含まれるブロモフェノールブルーがゲルから流れ出す寸前まで室温で約17時間泳動を行った（図1(1)参照：図中、1Dは一次元目の電気泳動方向を示す。以下同じ）。 (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 RNA sample for electrophoresis in the gel sample injection groove (width 1cm x depth 1cm x thickness 0.2cm) and at room temperature until the bromophenol blue contained in the sample flows out from the gel at a constant current of 10mA. (See FIG. 1 (1): In the figure, 1D indicates the first-dimensional electrophoresis direction. The same applies hereinafter).

一次元目の電気泳動が終わった後、ゲル板を泳動装置から外し、ゲル板全体を、疎水性透明フィルムであるポリ塩化ビニリデン製の食品包装用透明ラップフィルムで密着包囲した。蛍光テープの目印をラップフィルム上に付けてから、オートラジオグラフィーを行った（富士写真フィルム社の商品名：FUJI X-ray film RAを使用）。感光はインテンシファイイング・スクリーンを用いて-80℃で16時間〜24時間行った。ラップフィルム上の蛍光テープの目印と、感光したオートラジオグラムに印された蛍光テープ位置をあわせ、感光検出された50nt以下の低分子RNAを含む領域のゲルの部分（泳動レーンの幅は1cm）の表面に位置するラップフィルムに、薄板状部材として、縦15.5cm×横0.5cmに切断した、一方の面が疎水性面で他方の面が親水性面であるPharmacia Biotech社製の商品名：Gel Bond PAG filmの疎水性面を当接させ、カッターナイフを用いてゲルをラップフィルムとともに薄板状部材の周縁部に沿って切り込んだ。次に、薄板状部材を、切り込んだゲルの底面にその疎水性面が当接するようにゲルの下に潜り込ませ、薄板状部材を支持体にしてゲルを取り出した（図1(2)参照）。なお、ゲルの切り出し幅を泳動レーンの幅より狭くしたのは、後に行う、二次元目の電気泳動を始めてすぐに、ストリップ状に切り出した一次元目のゲル中に存在する50nt以下の低分子RNAを、二次元目のゲルに移動させることで、最終的に二次元目のゲル上で前記低分子RNAを再現性よく分離させるためであった。   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.

一次元目の電気泳動が終了する2時間前に、二次元目の電気泳動の準備を行った。なぜなら、ゲルは乾燥して壊れやすいものであるため、一次元目のゲルを切り出した後は、すぐにこれを二次元目の電気泳動を行うためのゲルと重合させることができる態勢にしておくことが好ましいからであった。二次元目の電気泳動を行うためのゲルとして、アクリルアミド濃度が22.0%で尿素濃度が7.0Mのスラブ状ポリアクリルアミドゲルを用いることにした。このゲルは、40%アクリルアミド溶液132ml、10×TBE 7.2ml、水7ml、尿素100gの組成で調製した。各成分を300mlビーカーに入れ、約1時間かけて尿素を完全に溶解させた。尿素が完全に溶解した後、得られた溶液を濾過してから脱気し、300mlビーカーに注いで泡立てない程度にスターラーで混ぜ、ここにN,N,N',N'-テトラメチルエチレンジアミン（TEMED）230μlと10%過硫酸アンモニウム1mlを加えて素早く混ぜた後、得られた溶液を、薄板状部材を用いてストリップ状に切り出した一次元目のゲルをセットした、二次元目の電気泳動に用いるガラス製ゲル板（縦47.5cm×横19.5cm×隙間0.2cm）に、一次元目のゲルの下に気泡が入らないように静かに流し込んで重合させて調製した。   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.

アクリルアミドの重合反応が終了してゲルが固まった後、ゲル板を泳動装置にセットして（少なくともこの段階に至るまでには一次元目のゲルの表面に残ったゲルとともに切り込まれたラップフィルムは除去済みである）、ゲル板上下のバッファー槽を0.3×TBEで満たし、ゲルの最上部に泳動状況識別用電気泳動用尿素バッファー（7M尿素，0.25%ブロモフェノールブルー，0.25%キシレンシアノール）を約100μlマウントし、10mA定電流、室温条件下、電気泳動用尿素バッファーに含まれるキシレンシアノールが一次元目のゲルから30cm下に移動するまで約24時間泳動を行った（図1(3)参照：図中、2Dは二次元目の電気泳動方向を示す。以下同じ）。二次元目の電気泳動が終了した後、一次元目の電気泳動が終了した時と同様の手順でオートラジオグラフィーを行った。その結果、二次元目のゲル上で多数の低分子RNAを分離することができた（図1(4)参照）。   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, a large number of small RNAs could be separated on the second-dimensional gel (see FIG. 1 (4)).

実験2：
embryonic stageの線虫（孵化前の線虫）から調製した試料と、mixed stageの線虫（卵から成虫に至るまでの幼虫期L1〜L4のいずれかの段階にある線虫が複数混在しているもの）から調製した試料を、それぞれ実験1と同様にして二次元電気泳動を行い、50nt以下の低分子RNAを二次元目のゲル上で分離した。結果をそれぞれ図2の(1)と(2)に示す（図中、1Dは一次元目の電気泳動方向を示し、2Dは二次元目の電気泳動方向を示す）。両者を比較すると互いに泳動像が異なり、それぞれのステージにおいて生体内における低分子RNAの発現状態が異なることがわかった。検出されたスポットは、embryonic stageに特異的なもの（例えば図2(1)で矢視したもの）が85個、mixed stageに特異的なもの（例えば図2(2)で矢視したもの）が51個、共通に見られるもの（例えば円で囲んだもの）が54個であった。こうして得られたスポットから、新規な50nt以下の低分子RNAの探索などを行うことができた。 Experiment 2:
Samples prepared from embryonic stage nematodes (pre-hatch nematodes) and mixed stage nematodes (larva stages L1 to L4 from egg to adult stage) 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.

本発明は、50nt以下の低分子RNAを簡便に再現性よく分離することができる二次元電気泳動法を用いた、その分析方法を提供することができる点において、産業上の利用可能性を有する。   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. .

実施例における本発明の二次元電気泳動法を用いた50nt以下の低分子RNAの分析方法の概略手順を示す図である。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. 同、embryonic stageの線虫の低分子RNAの二次元電気泳動像と、mixed stageの線虫の低分子RNAの二次元電気泳動像である。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)

二次元電気泳動法を用いた50ヌクレオチド以下の低分子RNAの分析方法であって、アクリルアミド濃度が12.5%〜13.5%のスラブ状ポリアクリルアミドゲルを用いて試料の一次元目の電気泳動を行った後、泳動レーン中の前記低分子RNAを含む領域のゲルをストリップ状に切り出す際、ゲル板全体を疎水性透明フィルムで密着包囲し、切り出すゲルの部分の形状と略同一形状であって、少なくとも一方の面に疎水性面を有する薄板状部材のいずれかの面を、切り出すゲルの部分の表面に位置する疎水性透明フィルムに当接させ、ゲルを疎水性透明フィルムとともに薄板状部材の周縁部に沿って切り込み、次に、薄板状部材を、切り込んだゲルの底面にその疎水性面が当接するようにゲルの下に潜り込ませ、薄板状部材を支持体にしてゲルを取り出し、このようにして切り出したゲルを二次元目の電気泳動を行うためのゲルと重合させた後、電気泳動を行うことを特徴とする分析方法。   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. 薄板状部材の疎水性面を疎水性透明フィルムに当接させ、ゲルを疎水性透明フィルムとともに薄板状部材の周縁部に沿って切り込むことを特徴とする請求項1記載の分析方法。   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. 少なくとも2種類の特定の条件下にある個体または細胞を用いて調製した試料の泳動パターンを比較することで、前記低分子RNAの発現特性を比較分析することを特徴とする請求項1または2記載の分析方法。   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. 請求項1乃至3のいずれかに記載の分析方法を行うためのキットであって、一次元目の電気泳動を行うためのゲル板とゲル形成用試薬、二次元目の電気泳動を行うためのゲル板とゲル形成用試薬、一次元目の電気泳動を行う際にゲルに試料注入溝を形成するための櫛、疎水性透明フィルム、少なくとも一方の面に疎水性面を有する薄板状部材を少なくとも含んでなることを特徴とするキット。   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.
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JPN6010050522, ＩＫＥＭＵＲＡ Ｔ, "Ｐｕｒｉｆｉｃａｔｉｏｎ ｏｆ ＲＮＡ Ｍｏｌｅｃｕｌｅｓ ｂｙ Ｇｅｌ Ｔｅｃｈｎｉｑｕｅｓ", ＭＥＴＨＯＤＳ ＩＮ ＥＮＺＹＭＯＬＯＧＹ, 1989, ｖｏｌ． １８０, ｐａｇｅｓ １４ − ２５ *
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