A high resolution RNA electrophoresis method

Abstract

This invention relates to a high-resolution RNA electrophoresis method, comprising the following steps: RNA pretreatment; gel preparation; electrophoresis; and gel imaging. The technical solution provided by this invention involves disrupting the secondary structure of RNA to form a uniform, irregular structure, followed by electrophoresis in a low-ion-concentration sodium borate buffer (SB buffer). The borate ions in the buffer cross-link agarose, reducing the porosity of the agarose gel and thus improving the separation of fragments smaller than 1000 bp, resulting in clear bands. This method eliminates the need for formaldehyde used in traditional methods, improving experimental safety. Furthermore, the low ion concentration of the electrophoresis buffer results in low current and minimal heat generation during electrophoresis, allowing for the use of higher voltages and shorter electrophoresis times, while also reducing experimental operating costs.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis, and specifically relates to a high-resolution RNA electrophoresis method. Background Technology

[0002] Gel electrophoresis is a common method for detecting the length of DNA and RNA fragments, including agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE). Agarose gel electrophoresis can separate nucleic acid fragments in the range of 0.1-25 kbp, while polyacrylamide gel electrophoresis is better at separating small fragments below 500 bp, with a maximum resolution of one base.

[0003] The negative logarithm (pKa) of the ionization equilibrium constant of hydrogen in the phosphate group of a nucleic acid backbone is approximately 6-7. At pH greater than 7, the hydrogen in the phosphate group ionizes, so nucleic acids carry a negative charge in neutral to alkaline pH conditions and will migrate from the negative electrode to the positive electrode in an electric field. The migration speed of nucleic acids in an electric field is mainly related to fragment length, but the structure of the nucleic acid also affects the migration speed. A prerequisite for determining fragment length in nucleic acid electrophoresis is that all nucleic acid fragments are linear, and within a certain length range, the migration speed is directly proportional to the fragment length. However, in reality, nucleic acids themselves fold and bend, forming higher-order structures. For example, double-stranded circular plasmid DNA can form supercoils, making its structure more compact and migrating faster during electrophoresis than straight-stranded DNA of the same length. Single-stranded RNA can form more complex structures, having a greater impact on migration speed. For these samples, a pretreatment step can be added before electrophoresis to convert the nucleic acids into linear straight chains, thus eliminating the influence of structure on migration speed. For example, before electrophoresis, restriction endonucleases are used to cut circular DNA into linear DNA, or denaturants are added to prevent RNA from forming intramolecular base pairs.

[0004] The two main components of gel electrophoresis are the gel medium and the electrophoresis buffer. The pore size of the gel medium determines the size of the separable fragments. The electrophoresis buffer transmits the electric field and maintains pH stability. Commonly used electrophoresis buffers for agarose gel electrophoresis are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA), formulations that have remained unchanged since the early days of molecular biology. Researchers at the time may not have systematically optimized the components, and coupled with a lack of understanding of some underlying principles, while these methods were effective, they were not the most perfect.

[0005] Currently, the most common method for RNA electrophoresis in laboratories is formaldehyde MOPS agarose gel electrophoresis. This method requires denaturing RNA into single strands using formaldehyde and formamide, and 2% formaldehyde is added during gel preparation, so it must be performed in a fume hood. The MOPS buffer used is pH 7, not the usual pH 8, to avoid alkaline degradation of RNA. However, the buffer cannot be acidic, because nucleic acids are uncharged at acidic pH and cannot move in an electric field. There is also an electrophoresis method that uses glyoxal to denature RNA, but this method has more complex reagents and procedures.

[0006] Because formaldehyde MOPS agarose gel electrophoresis uses toxic reagents and is cumbersome, TAE agarose gel electrophoresis is usually used directly to detect RNA, without denaturing the RNA before electrophoresis. However, TAE agarose gel electrophoresis is not very effective for separating RNA because the high ion concentration in the TAE electrophoresis buffer allows RNA to form secondary structures, often resulting in diffusion or tailing of even undegraded RNA. This has little impact on animal RNA, which typically has only two main bands; even with slight diffusion due to secondary structures, the 18S and 28S rRNA bands can still be distinguished. However, plant leaf total RNA contains a variety of fragments, and the overlapping of these fragments can easily create the illusion of RNA degradation. Summary of the Invention

[0007] Existing technologies are not very accurate, involve complicated steps, require toxic reagents, have long electrophoresis times, and are relatively expensive.

[0008] To address the aforementioned technical problems, this invention discloses a high-resolution RNA electrophoresis method. The method involves denaturing RNA at a low ion concentration, followed by electrophoresis in a low-ion-concentration sodium borate buffer (SB buffer), following these steps:

[0009] A high-resolution RNA electrophoresis method includes the following steps:

[0010] Step 1: RNA Pretreatment

[0011] There are two options for RNA denaturation pretreatment.

[0012] Option 1: Directly heat RNA dissolved in pure water;

[0013] Option 2: Mix RNA with formamide and then heat;

[0014] Step 2: Glue preparation

[0015] Add agarose to low-ionic-concentration electrophoresis buffer, heat to melt and mix well, then pour into a mold and wait for it to solidify;

[0016] Step 3: Electrophoresis

[0017] Add low-ion-concentration electrophoresis buffer to the electrophoresis tank, put the solidified gel into the electrophoresis tank so that the electrophoresis buffer just covers the gel, and then load the sample for electrophoresis.

[0018] Step 4: Gel Imaging

[0019] Observe and photograph using a gel imaging system.

[0020] Preferably, in step one, the temperature conditions for both RNA pretreatment methods are heating at 65°C for 5 minutes, followed by immediate rapid cooling on ice.

[0021] Preferably, in step one, in RNA pretreatment option 2, the volume of formamide is 1.5 times that of the RNA.

[0022] Preferably, in steps two and three, the electrophoresis solution used is a low-ion-concentration electrophoresis solution, including but not limited to sodium borate electrophoresis solution (SB), lithium borate electrophoresis solution (LB), and lithium acetate borate electrophoresis solution (LAB). These electrophoresis solutions have an ion concentration of less than 10 mM, while common electrophoresis solutions, such as TAE, TBE, and MOPS electrophoresis solutions, have an ion concentration greater than 50 mM.

[0023] Preferably, in step two, the SB electrophoresis solution is prepared by mixing 200mM sodium hydroxide and 720mM boric acid and then diluting the mixture 20 times, resulting in a pH of 7.5 for the diluted electrophoresis solution.

[0024] Preferably, the high-resolution RNA electrophoresis method further includes a staining step, with two options: Option 1: After heating, melting, and mixing in step two and pouring the mixture into a mold, stain with Good View nucleic acid dye; Option 2: After electrophoresis in step three, stain with SYBR gold dye.

[0025] More preferably, in step two, in option 1, the dilution ratio of the Good View nucleic acid dye is 10000:1, 5000:1, 10000:1, or 100000:1.

[0026] Preferably, in step three, there are three options for RNA loading methods: Option 1: RNA is mixed with 10× loading buffer at a ratio of 9:1 and then loaded; Option 2: RNA is mixed with 50% glycerol at a ratio of 9:1 and then loaded; Option 3: RNA is directly loaded with a mixture of formamide at a ratio of 4:6.

[0027] More preferably, in step three, option 1, the 10× loading buffer is prepared in the following proportions: 50% glycerol, 5 mM Orange G, and 10 mM EDTA.

[0028] Preferably, in the above steps, the RNA is in a solution with a low ion concentration, thereby ensuring that the RNA does not form a complex secondary structure without the use of toxic denaturing agents such as formaldehyde; the low ion concentration refers to a cation concentration of no more than 10 mM, including pure water.

[0029] The beneficial effects of this invention are:

[0030] 1. This method destroys the secondary structure of RNA by heating at low ion concentrations, and after cooling, a uniform irregular structure is formed, which makes the electrophoretic velocity of all molecules of the same RNA consistent, thus forming clear bands.

[0031] 2. This method does not require the use of formaldehyde, which improves the safety of the experiment.

[0032] 3. The low salt concentration of the electrophoresis solution in this method results in a small current and low heat generation during electrophoresis, allowing for the use of higher voltages and shorter electrophoresis times.

[0033] 4. This method uses a low-salt-concentration electrophoresis buffer, which also reduces experimental costs. Based on 1L of electrophoresis buffer per electrophoresis, the cost of sodium borate electrophoresis buffer is 0.12 yuan / L, which is one-eighth of TAE, one-tenth of TBE, and one-twentieth of MOPS buffer.

[0034] 5. This method uses SYBR gold as a nucleic acid dye and modifies the process to staining after electrophoresis, enabling the detection of total RNA as low as 10 ng.

[0035] 6. The electrophoresis buffer used in this method contains boric acid, which can cross-link agarose, greatly improving the resolution of RNA electrophoresis. Attached Figure Description

[0036] Figure 1 The images show the electrophoretic effects of three different electrophoresis solutions on RNA.

[0037] Figure 2 This is a schematic diagram of RNA electrophoresis in formaldehyde denaturing gel, TAE, and SB agarose gel.

[0038] Figure 3 This is a schematic diagram illustrating the minimum detection limit of SB agarose gel electrophoresis. Detailed Implementation

[0039] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] Example 1

[0041] I. RNA pretreatment: RNA samples from Arabidopsis thaliana leaves were extracted using the Trizol method at a concentration of approximately 440 ng / μL. 882 ng, 617 ng, 441 ng, 176 ng, and 88 ng of RNA were taken, and water was added to make up to 4 μL. The mixture was then mixed with 6 μL of formamide. After heating the RNA at 65 °C for 5 min, it was immediately placed on ice for rapid cooling.

[0042] II. Gel preparation: Weigh 0.3g of agarose and add it to 30mL of 1×SB electrophoresis buffer. After heating and melting, add Good View nucleic acid dye (dilution ratio 10000:1), mix well, pour into the mold, and wait for solidification.

[0043] III. Electrophoresis: Add 1×SB electrophoresis buffer to the electrophoresis tank, place the gel into the electrophoresis tank so that the electrophoresis buffer just covers the gel, mix 9μL of the heated denatured RNA sample with 1μL of 10× loading buffer and load the sample, set the electrophoresis voltage to 100V and perform electrophoresis.

[0044] IV. Gel imaging: After electrophoresis, observe and photograph the gel using a gel imaging system.

[0045] V. Electrophoresis Image Analysis: The RNA gel electrophoresis images obtained using this embodiment (… Figure 1 The left side (1×SB) can identify 11 bands in total plant RNA. This example also shows that, using GoodView as a nucleic acid dye, the limit of RNA detection is 100 ng.

[0046] The majority of total RNA in Arabidopsis leaves is ribosomal RNA, including eukaryotic 28S rRNA and 18S rRNA in the cytoplasm. Chloroplasts and mitochondria also have their own ribosomal RNA. Chloroplast ribosomal RNA is mainly 23S rRNA and 16S rRNA. The 23S rRNA in chloroplasts contains two hidden breaks, making it prone to fragmentation into five different lengths. Plant total RNA electrophoresis can reveal up to 11 bands, which cannot be separated by conventional non-denaturing gel electrophoresis. Figure 1 (Right side 1×TAE). The amount of mitochondrial RNA is so small that it cannot be seen in the electrophoresis image.

[0047] Example 2

[0048] This example demonstrates that using SYBR gold as a nucleic acid dye, trace amounts of RNA as low as 10 ng can be detected.

[0049] I. RNA Pretreatment: RNA samples from Arabidopsis thaliana leaves were extracted using the Trizol method at a concentration of approximately 440 ng / μL. 200 ng, 100 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, and 0 ng of RNA were taken respectively, and water was added to make up to 4 μL. Then, the RNA was mixed with 6 μL of formamide. After heating the RNA at 65 °C for 5 min, it was immediately placed on ice for rapid cooling.

[0050] II. Gel preparation: Weigh 0.3g of agarose and add it to 30mL of 1×SB electrophoresis buffer. Heat until melted, then pour into a mold and wait for it to solidify.

[0051] III. Electrophoresis: Add 1×SB electrophoresis buffer to the electrophoresis tank, place the gel in the electrophoresis tank so that the electrophoresis buffer just covers the gel, mix the heated denatured RNA sample with 1μl of 10× loading buffer and load the sample, set the electrophoresis voltage to 100V and perform electrophoresis, and stain with SYBR gold after electrophoresis.

[0052] IV. Gel imaging: After electrophoresis, observe and photograph the gel using a gel imaging system.

[0053] V. Electrophoretic Image Analysis: Electrophoretic Image Display ( Figure 3 This method, combined with the more sensitive SYBR gold nucleic acid dye, can detect RNA as low as 10 ng.

[0054] In Example 2, it is important to note that the order of electrophoresis and staining is crucial in microRNA electrophoresis detection. SYBR gold must be added for staining only after electrophoresis. If SYBR gold is added directly to the gel for electrophoresis, the RNA bands will be distorted and severely tailed. This may be because the binding of SYBR gold to RNA alters the RNA structure, or because SYBR gold causes uneven local electric field distribution, leading to band distortion.

[0055] like Figure 1 As shown, from left to right, these are electrophoresis results of RNA using SB, TBE, and TAE electrophoresis solutions in Example 1. The first image on the left is the electrophoresis diagram of this invention. It can be seen that in SB electrophoresis, RNA fragments from chloroplasts can be separated well, and 100nt and 150nt fragments can also be separated. The second and third images on the left are comparison images. In TAE electrophoresis, RNA from chloroplasts is blurred due to insufficient separation. Because the resolution for small fragments is insufficient, 100nt and 150nt fragments are compressed into one band.

[0056] like Figure 2The diagram shows the electrophoresis of RNA in formaldehyde denaturing gel, TAE, and SB gel. The diagrams show the electrophoresis of three RNA molecules of the same type; the first and second from the left are comparative images, and the third from the left is an example of the results of this invention. In TAE electrophoresis, RNA forms various secondary structures, which have different effects on its migration speed. Even undegraded RNA often exhibits diffusion or tailing phenomena. However, in formaldehyde denaturing gel and SB electrophoresis, RNA does not form secondary structures, and the structure of RNA has less impact on its migration speed, resulting in clear bands.

[0057] like Figure 3 The diagram illustrates the minimum detection limit of SB agarose gel electrophoresis. Typically, RNA agarose gel electrophoresis requires 1 μg of total RNA. Some sophisticated experiments cannot provide this much RNA and require methods like capillary electrophoresis for detection. With slight modifications, the method of this invention can be used to detect trace amounts of RNA. SYBR gold's sensitivity is 25 times that of ethidium bromide. By using SYBR gold as the nucleic acid dye and modifying the staining process to post-electrophoresis, the method of this invention can detect as little as 10 ng of total RNA, approaching the minimum detection limit of some capillary electrophoresis instruments.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A high-resolution RNA electrophoresis method, comprising the following steps: Step 1: RNA Pretreatment There are two options for RNA denaturation pretreatment. Option 1: Directly heat RNA dissolved in pure water, and immediately place it on ice to cool it rapidly after heating; Option 2: Mix RNA with formamide and heat, then immediately place on ice to cool rapidly. Step 2: Glue preparation Add agarose to low-ionic-concentration electrophoresis buffer, heat to melt and mix well, then pour into a mold and wait for it to solidify; Step 3: Electrophoresis Add low-ion-concentration electrophoresis buffer to the electrophoresis tank, put the solidified gel into the electrophoresis tank so that the electrophoresis buffer just covers the gel, and then load the sample for electrophoresis. Step 4: Gel Imaging Observe and photograph using a gel imaging system; The low-ion-concentration electrophoresis solution is one of sodium borate electrophoresis solution, lithium borate electrophoresis solution, and lithium acetate-boric acid electrophoresis solution; the low-ion-concentration means that the cation concentration does not exceed 10 mM. Throughout all steps, the RNA is kept in a solution with low ion concentration, thus ensuring that the RNA does not form complex secondary structures without the use of formaldehyde.

2. The high-resolution RNA electrophoresis method according to claim 1, characterized in that, In step one, the temperature conditions for both RNA pretreatment methods are heating at 65°C for 5 minutes, followed by immediate rapid cooling on ice.

3. The RNA electrophoresis method according to claim 1, characterized in that, In step one, in RNA pretreatment option 2, the volume of formamide is 1.5 times that of the RNA.

4. The high-resolution RNA electrophoresis method according to claim 1, characterized in that, The SB electrophoresis buffer is prepared by mixing 200mM sodium hydroxide and 720mM boric acid and then diluting it 20 times. The pH of the diluted electrophoresis buffer is 7.

5.

5. The high-resolution RNA electrophoresis method according to claim 1, characterized in that, The high-resolution RNA electrophoresis method also includes a staining step, with two options: Option 1: After heating, melting, and mixing in step 2, pour the mixture into a mold and then stain with GoodView nucleic acid dye; Option 2: After electrophoresis in step 3, stain with SYBR Gold dye.

6. The high-resolution RNA electrophoresis method according to claim 5, characterized in that, In step two, the GoodView nucleic acid dye used in option 1 is diluted at a ratio of 5000:1, 10000:1, or 100000:

1.

7. The high-resolution RNA electrophoresis method according to claim 1, characterized in that, In step three, there are three options for RNA loading: Option 1: RNA is mixed with 10× loading buffer at a volume ratio of 9:1 and then loaded; Option 2: RNA is mixed with 50% glycerol at a volume ratio of 9:1 and then loaded; Option 3: RNA is directly loaded with a mixture of formamide at a volume ratio of 4:

6.

8. The high-resolution RNA electrophoresis method according to claim 7, characterized in that, In step 3, option 1, the 10× loading buffer is prepared with the following ratio: 50% glycerol, 5mM Orange G, and 10mM EDTA.

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