WO2006073175A1

WO2006073175A1 - Method of analyzing low-molecular weight rna

Info

Publication number: WO2006073175A1
Application number: PCT/JP2006/300078
Authority: WO
Inventors: Chisato Ushida
Original assignee: Hirosaki University
Priority date: 2005-01-07
Filing date: 2006-01-06
Publication date: 2006-07-13
Also published as: JP4599555B2; JP2006189359A

Abstract

[PROBLEMS] To provide an analysis method with the use of two dimensional electrophoresis whereby a low-molecular weight RNA of 50 nt or less can be conveniently separated at a high reproducibility. [MEANS FOR SOLVING PROBLEMS] An analysis method characterized by comprising: subjecting a sample to the first dimensional electrophoresis by using a slab polyacrylamide gel having an acrylamide concentration of from 12.5% to 13.5%; in cutting the gel in the region containing the low-molecular weight RNA in the electrophoretic lane into strips, tightly covering the whole gel plate with a hydrophobic transparent film, bringing some face of a thin plate member, which is roughly the same in shape as the shape of the gel part to be cut out and has a hydrophobic face as at least one face thereof, into contact with the hydrophobic transparent film located on the surface of the gel part to be cut out, cutting the gel together with the hydrophobic transparent film along the periphery of the thin plate member, sliding the thin plate member under the gel so that the hydrophobic face of the thin plate member comes into contact with the gel bottom, then cutting out the gel while using the thin plate member as a support; polymerizing the gel thus cut out with a gel for the second dimensional electrophoresis and then carrying out the electrophoresis.

Description

Specification

Analysis method for small RNA

Technical field

[0001] The present invention relates to a method for analyzing small RNA using two-dimensional electrophoresis.

Background art

[0002] In recent years, the discovery of RNA interference (RNAi) and the fact that 22-26 nucleotide (nt) small RNAs have been found to be deeply involved in its mechanism of action have been revealed. The existence of a large number of small RNAs or non-coding RNAs (ncRNAs) in cells and the functions of small RNAs are elucidating. 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 energetically, a method that can separate and analyze small RNAs with excellent reproducibility is indispensable. Therefore, such a method is awaited in the field of research. One of the potential methods for analyzing small RNAs is the two-dimensional electrophoresis method. This method has already been proposed in Non-Patent Document 1. However, in Non-Patent Document 1, it has been confirmed that a mixture of artificially synthesized les and a few small RNAs of 20 or more nucleotides can be separated by two-dimensional electrophoresis. However, it is not yet possible to actually isolate and analyze a mixture of naturally occurring small RNAs. Also, in Non-Patent Document 1, the details of specific methods for performing two-dimensional electrophoresis are not disclosed. In addition to the method proposed in Non-Patent Document 1, there is a method proposed in Non-Patent Document 2, for example, 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 the separation, Skilled technology is required and not everyone can do it easily.

Non-patent or 1: Two-dimensional rractionation or complex mixture of oligonucleotides in microRNA size range: Hideaki Shiraishi, 2004 Oxford University Press, Nucleic A cids Symposium Series No. 48, 293-294

Non-Patent Document 2: Purification of RNA Molecules by Gel techniques: Toshimichi Ikemura, Methods In Enzymology, Vol.180, ppl4-25 (1989)

Disclosure of the invention

Problems to be solved by the invention

[0003] Therefore, an object of the present invention is to 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 high reproducibility.

Means for solving the problem

[0004] As a result of intensive research in view of the above points, the present inventor has performed the first-dimensional electrophoresis when analyzing a low-molecular-weight RNA of 50 nt or less using a two-dimensional electrophoresis method. The second-dimensional gel is identified by identifying the gel to be used in the electrophoresis, and after the first-dimensional electrophoresis, by identifying the method for cutting out the gel containing the low-molecular-weight RNA in the electrophoresis lane. From the above, it has been found that the low molecular weight RNA can be easily and reproducibly separated.

[0005] A method for analyzing a low molecular weight RNA of 50 nt or less using the two-dimensional electrophoresis method of the present invention based on the above findings is a slab having an acrylamide concentration of 12.5% to 13.5% as described in claim 1. After performing the first-dimensional electrophoresis of the sample using a glass polyacrylamide gel, when the gel of the region containing the low-molecular RNA in the electrophoresis lane is cut into a strip shape, the entire gel plate is covered with a hydrophobic transparent film. Hydrophobic transparent located on the surface of the gel part to be cut out, either side of the thin plate-like member that is close to the shape of the gel part to be enclosed and cut out and has a hydrophobic surface on at least one side Abut the film, cut the gel along with the hydrophobic transparent film along the periphery of the thin plate member, and then place the thin plate member under the gel so that the hydrophobic surface is in contact with the bottom surface of the cut gel. Dive into The gel is taken out using the thin plate member as a support, and the gel cut out in this way is polymerized with the gel for performing the second-dimensional electrophoresis, and then the electrophoresis is performed. .

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 is combined with the hydrophobic transparent film. It cuts along the peripheral part of a thin plate-shaped member, It is characterized by the above-mentioned.

The analysis method according to claim 3 compares the migration patterns of samples prepared using individuals or cells under at least two specific conditions in the analysis method according to claim 2 or 2. Thus, the expression characteristics of the low-molecular 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 during first-dimensional electrophoresis, hydrophobic transparent film, on at least one surface It comprises at least a thin plate-like member having a hydrophobic surface. The invention's effect

[0006] 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.

Brief Description of Drawings

[0007] FIG. 1 is a diagram showing a schematic procedure of a method for analyzing a small RNA of 50 nt or less using the two-dimensional electrophoresis method of the present invention in Examples.

[Fig.2] Two-dimensional electrophoresis images of embryonic stage nematode small RNA and two-dimensional electrophoretic images of mixed stage nematode small RNA.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] The method for analyzing low-molecular-weight RNA of 50 nt or less using the two-dimensional electrophoresis method of the present invention uses a slab-like polyacrylamide gel having an atalylamide concentration of 12.5% to 13.5% to perform the first-dimensional electrolysis of a sample After the electrophoresis, when the gel in the region containing the low molecular weight RNA in the electrophoresis lane is cut out in a strip shape, the entire gel plate is closely enclosed with a hydrophobic transparent film, and the shape of the gel part to be cut out is almost the same. One of the thin plate-like members that has a shape and has a hydrophobic surface on at least one surface is brought into contact with a hydrophobic transparent film located on the surface of the gel part to be cut out, thereby making the gel hydrophobic and transparent. Cut along the peripheral edge of the thin plate member together with the film, and then sink the thin plate member under the gel so that the hydrophobic surface is in contact with the bottom surface of the cut gel, and use the thin plate member as a support. Take out the gel In this way The gel cut out in this manner is polymerized with the gel for performing the second-dimensional electrophoresis, and then electrophoresed.

[0009] 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 for identifying the gel type as polyacrylamide gel is that it has excellent resolution for a mixture of small RNAs of less than 50 nt to be analyzed. The acrylamide concentration is specified as 12.5% to 13.5% because if the concentration falls below 12.5%, it becomes a condition that improves the resolution for a mixture of RNA larger than 50M, and conversely it is as low as 50 nt or less. This is because the resolution of the molecular RNA mixture decreases. On the other hand, when the concentration exceeds 13.5%, when performing second-dimensional electrophoresis under the conditions described later, the difference from the acrylamide concentration of the gel used for second-dimensional electrophoresis is reduced. This is because the resolution of RNA spots obtained after the second-dimensional electrophoresis deteriorates, making it difficult to identify individual spots.

[0010] 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 into a strip shape, the entire gel plate is treated with a hydrophobic transparent film. The surface of the gel part to be cut out is one surface of the thin plate-like member which is substantially the same shape as the part of the gel part to be tightly enclosed and cut and has a hydrophobic surface on at least one side. The gel is cut along the peripheral edge of the thin plate member together with the hydrophobic transparent film, and then the thin plate member is cut so that the hydrophobic surface comes into contact with the bottom surface of the cut gel. Under the gel, the gel is taken out using the thin plate member as a support. When cutting the gel into strips, the length of the cut is preferably shorter than the width of the gel used for the second-dimensional electrophoresis, and the cut width is preferably smaller than the width of the electrophoresis lane. By performing as described above, it is possible to easily and accurately cut out a very thin strip-shaped gel with a cutting length of 10 to 20 cm and a cutting width of 0.3 to 0.7 cm. It becomes.

[0011] Examples of the hydrophobic transparent film used for tightly enclosing the entire gel plate include a transparent wrap film for food packaging made of polyvinylidene chloride. If a hydrophilic transparent film such as cellophane is used instead of the hydrophobic transparent film, the film softens and expands with moisture from the electrophoresis buffer remaining on the gel surface. As a result, it becomes difficult to tightly enclose the entire gel plate, resulting in a problem that the gel cannot be cut out accurately.

[0012] The thin plate-like member having a hydrophobic surface on at least one surface may be any member as long as it has a hydrophobic surface that is necessary when the cut gel is taken out later. However, it is preferable that the surface of the thin plate-like member to be brought into contact with the hydrophobic transparent film used for tightly enclosing the entire gel plate has an affinity for the hydrophobic transparent film (polysalt as the hydrophobic transparent film). When using a transparent wrap film for food packaging made of 匕 vinylidene, the hydrophobic surface of the thin plate member has 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-like member, slippage between the hydrophobic transparent film and the thin plate-like member is suppressed, and a gel having a straight cut is cut out easily and accurately. Because it becomes possible. Further, 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 the thin plate-like member is used as a support to take out the gel. 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 to remove the gel, the water present on the gel surface interferes with the thin plate-like member. When it becomes difficult to accurately dive under the gel into which the gel is cut, it becomes difficult to set the gel on the gel plate used for the second-dimensional electrophoresis.

[0013] 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 6.0 \ 4-8.0 \ 4, for example. Les. The reason why the gel type is polyacrylamide gel is that it is excellent in the resolution of a mixture of small RNAs of 50 nt or less to be analyzed. It is preferable to set the acrylamide concentration to 21.0% to 23.0%. If the concentration force is less than ¾1.0%, 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 eye electrophoresis deteriorates, making it difficult to identify individual spots. On the other hand, if the concentration exceeds 23.0%, the resolution of RNA near 50 nt will be poor, and the gel will be easily broken, making it difficult to handle. The urea concentration is preferably 6.0M to 8.0M because of the difference in the secondary structure of small RNAs. This is because the influence on the second-dimensional electrophoresis can be eliminated. By adding urea with a concentration of 6.0M-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.

[0014] It should be noted that a method for preparing a sample or gel containing a low molecular weight RNA of 50 nt or less and a method for electrophoresis are carried out by a method known per se (if necessary, "Polyacrylamide Gel Electrophoresis Molecular"). lomng, a laboratory manual, second edition: J. Sambrook, EF F itsch and T. Maniatis (1989), pp. 6.36-6.48, Cold Spring Harbor Laboratory Press, New York.

[0015] According to the present invention, low molecular weight RNA of 50 nt or less (approximately 15 nt to 50 nt) can be easily and reproducibly separated on the second-dimensional gel. Therefore, at least some small RNAs of less than 50 nt can be obtained as high-purity as each spot on the gel, thus facilitating their sequencing and thus new 50 nt or less Efficient search for small RNAs. In addition, two-dimensional electrophoresis is performed on samples prepared using individuals or cells under at least two specific conditions, and the expression characteristics of small RNAs are compared and analyzed by comparing the migration patterns. Therefore, it is easy to clarify what kind of small RNAs are involved in higher life phenomena such as aging, behavior, neurogenesis, and embryogenesis in multicellular organisms.

[0016] The method for analyzing low molecular weight RNA of 50 nt or less using the two-dimensional electrophoresis method of the present invention comprises a gel plate and a gel-forming reagent for performing first-order electrophoresis, and a second-dimensional electrophoresis. Gel plate and gel forming reagent, comb for forming sample injection groove in gel when performing first-dimensional electrophoresis, hydrophobic transparent film, at least one surface has hydrophobic surface By using a kit comprising at least a thin plate-like member, anyone can easily perform 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 components shown in Table 1 below to a total amount of 213 ml with sterile water. [0017] [Table 1]

[0018] Examples of the gel forming reagent for performing the second-dimensional electrophoresis include an acrylamide solution prepared by mixing the components shown in Table 2 below to a total volume of 240 ml with sterile water.

[0019] [Table 2]

[0020] As a comb for forming a sample injection groove in the gel when performing the first-dimensional electrophoresis, for example, there are 10 teeth of 1 cm in length lcm x lcm in width at intervals of 0.2 cm, and the other in the other. An example is a Teflon (registered trademark) board with no teeth and a thickness of 0.2 cm. Examples of the hydrophobic transparent film include a transparent wrap film for food packaging made of polyvinyl chloride vinylidene. 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 used in Examples described later. The product name: Gel Bond PAG film manufactured by Pharmacia Biotech, which has a hydrophobic surface and the other surface is a hydrophilic surface.

Example

Hereinafter, the present invention will be described in detail by examples using nematodes (£.), But the present invention should not be construed as being limited to the following description.

[0022] Experiment 1:

(Preparation of RNA sample for electrophoresis)

Nematode 10g was treated with TRIzol reagent (Invitrogen) 10ml to obtain 0.5mg of crude RNA extract . This was fractionated by the stepwise 2-propanol precipitation method to obtain 5 xg of a fraction containing RNA below lOOnt. To label the 3 'end of RNA with a radioisotope, 2.5 μg of this RNA fraction, 6.5 μl of ³² P-pCp (ll lTBqAnmol), 20 unit of T4 RNA ligase (Takara Shuzo), final concentration Add 50 mM Tris-HCl (pH 7.5), lOmM MgCl, lOmM DTT, ImM ATP

2

In addition, the reaction was carried out at 4 ° C for 16 to 20 hours with sterilized water. Sterilize 5 μ 1 of glycerol buffer for electrophoresis (containing 50% glycerol, 0.25% bromophenol blue, and 0.25% xylene cyanol) into 15 μ 1 of the RNA fraction labeled at the 3 ′ end in this way. 5 μl of water was added to prepare an RNA sample for electrophoresis.

[0023] (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. That is, 40% acrylamide solution (Acrylic amide 380g and Ν, Ν'-methylenebisacrylamide 20g dissolved in water to 1000ml) 70ml, 10X TBE (Tris base 108g, boric acid 55g, 0.5M EDTA 40ml, pH 8.0) Add 6 ml and water 135 ml to a 300 ml beaker and mix with a stirrer to the extent that it does not foam, add TEMED230 μ 1 and 2 ml of 10% ammonium persulfate to this, quickly mix, and set up the resulting solution Poured into a glass gel plate (length 47.5 cm x width 19.5 cm x gap 0.2 cm). After the polymerization reaction of acrylamide was completed and the gel was solidified, the gel plate was set in the electrophoresis apparatus and pre-energized for 1 hour at 1000 V (constant voltage). Electrophoresis was performed using 0.3 X TBE as the electrophoresis buffer and a heat sink attached to the gel plate. After pre-energization, place the RNA sample for electrophoresis into the gel sample injection groove (width lcm x depth lcm x thickness 0.2cm) and immediately before bromphenol blue contained in the sample flows out of the gel at a constant current of 10mA. Electrophoresis was performed for about 17 hours at room temperature (see FIG. 1 (1): In the figure, 1D represents the first-dimensional electrophoresis direction; the same applies hereinafter).

[0024] After the first-dimensional electrophoresis is completed, the gel plate is removed from the electrophoresis apparatus, and the entire gel plate is closely adhered with a transparent wrap film for food packaging made of polysalt vinylidene, which is a hydrophobic transparent film. Besieged. After applying the fluorescent tape mark on the wrap film, autoradiography was performed (using Fuji Photo Film's product name: FUJI X-ray film RA). Photosensitivity was carried out at -80 ° C for 16-24 hours using an Intensive Eyeing 'screen. Fluorescent tape mark on the wrapping film and fluorescent tape stamped on the exposed autoradiogram 15.5 cm x 0.5 in width as a thin plate on a lap film located on the surface of the gel part (swimming lane width is 1 cm) of the region containing a small RNA of 50 nt or less that is photodetected and aligned. Product name: Gel Bond PAG film made by Pharm acia Biotech, which is cut into cm, one side is hydrophobic and the other side is hydrophilic. The gel was cut along with the wrap film along the peripheral edge of the thin plate member. 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 width of the cut out of the gel was made narrower than the width of the electrophoresis lane because the low-order 50 nt or less present in the first-dimensional gel cut into strips immediately after the second-dimensional electrophoretic movement performed later was started. This is because the low molecular weight RNA is finally separated on the second-dimensional gel with high reproducibility by moving the molecular RNA to the second-dimensional gel.

[0025] Two hours before the completion of the first-dimensional electrophoresis, preparation for the second-dimensional electrophoresis was made. Because the gel is dry and fragile, immediately after cutting out the first dimension gel, it is ready to polymerize with the gel for second dimension electrophoresis. This is because it is preferable to keep it. As a gel for the second-dimensional electrophoresis, a slab-like polyacrylamide gel with an acrylamide concentration of 22.0% and a urea concentration of S7.0M was used. This gel was prepared with a composition of 132 ml of 40% acrylamide solution, 7.2 ml of 10 X 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 solution obtained is filtered and degassed, poured into a 300 ml beaker and mixed with a stirrer to the extent that it does not foam, and に, Ν, Ν ', Ν'-tetramethylethylene is added here. After adding jamine (TEMED) 230 μl and 10% ammonium persulfate lml and mixing rapidly, the resulting solution was set in a first-dimensional gel that was cut into strips using a thin plate. Prepared by gently pouring into a glass gel plate (vertical 47.5 cm X horizontal 19.5 cm X gap 0.2 cm) used for the second-dimensional electrophoresis so that no bubbles enter under the first-dimensional gel. .

[0026] After the acrylamide polymerization reaction is completed and the gel is solidified, the gel plate is set in the electrophoresis apparatus (at least by this stage, the gel is cut with the gel remaining on the surface of the first dimension gel. The wrap film has been removed), and the buffer baths above and below the gel plate are 0.3 XT Filled with BE, mounted about 100 μΐ of urea buffer (7M urea, 0.25% bromophenol blue, 0.25% xylene cyanol) for electrophoresis identification at the top of the gel, 10 mA constant current, room temperature conditions Electrophoresis was performed for about 24 hours until xylene cyanol contained in the urea buffer for electrophoresis migrated 30 cm below the -dimensional gel (see Fig. 1 (3): In the figure, 2D represents the second-dimensional electricity The direction of electrophoresis is shown. 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)).

[0027] Experiment 2:

Samples prepared from embryonic stage nematodes (pre-hatch nematodes) and mixed stage nematodes (larva stages from eggs to adults) Samples prepared from 2) were subjected to two-dimensional electrophoresis in the same manner as in Experiment 1, and low-molecular-weight RNA of 50 nt or less was 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 that the expression state of small RNA in vivo was different at each stage. There are 85 spots detected that are specific to the embryonic stage (for example, those shown by arrows in Fig. 2 (1)), and those 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, circled). From the spots thus obtained, we were able to search for novel low molecular RNAs of 5 Ont or less.

Industrial applicability

[0028] The present invention is industrially applicable in that it can provide an analysis method using a two-dimensional electrophoresis method capable of easily and reproducibly separating low molecular weight RNA of 50 nt or less. Have potential.

Claims

The scope of the claims

[1] A method for analyzing small RNAs of 50 nucleotides or less using two-dimensional electrophoresis, and using a slab-like polyacrylamide gel with an acrylamide concentration of 12.5% to 13.5%, After cutting, when the gel in the region containing the low molecular weight RNA in the electrophoresis lane is cut out in a strip shape, the entire gel plate is tightly surrounded with a hydrophobic transparent film, and the shape of the gel portion to be cut out is approximately the same shape. Any one of the thin plate-like members having a hydrophobic surface on at least one surface is brought into contact with the hydrophobic transparent film located on the surface of the gel part to be cut out, and the gel is combined with the hydrophobic transparent film. Cut along the peripheral edge of the thin plate-like member, and then sink the thin plate-like member under the gel so that its hydrophobic surface is in contact with the bottom surface of the cut gel, and use the thin plate-like member as a support. Take An analysis method characterized by performing electrophoresis after polymerizing the gel cut out in this way and the gel for performing second-dimensional 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] A feature of the present invention is to compare and analyze the expression characteristics of the small RNA by comparing the swimming patterns of samples prepared using individuals or cells under at least two specific conditions. Item analysis method according to 2.

[4] A kit for performing the analysis method according to any one of claims 1 to 3, wherein the gel plate and the gel-forming reagent for performing the first-dimensional electrophoresis, and the second-dimensional electrophoresis are performed. Gel plate and gel forming reagent, comb for forming a sample injection groove in the gel during the first dimensional electrophoresis, hydrophobic transparent film, thin plate having a hydrophobic surface on at least one surface A kit comprising at least a shaped member.