A method for pre-cooling an agarose gel electrophoresis buffer for RNA integrity detection

Abstract

A method for pre-cooling an agarose gel electrophoresis buffer for RNA integrity detection, which belongs to the technical field of molecular biology experiments. TAE buffer and / or TBE buffer are placed in a clean and sealed container, and the container containing the buffer is placed in an ice bath environment for pre-cooling. The pre-cooling time is greater than or equal to 3 hours. The buffer subjected to deep pre-cooling is used to prepare an agarose gel. After the gel solidifies, it is poured into an electrophoresis tank. The buffer subjected to deep pre-cooling is injected into the electrophoresis tank, and the liquid surface needs to completely immerse the gel. Then, the RNA sample is mixed with the loading buffer and spotted into the sample well. The electrophoresis tank is placed in an environment with a temperature of 20-25 DEG C, and the power is turned on for electrophoresis. After the electrophoresis is completed, the gel is taken out, stained with nucleic acid, and observed and imaged using an ultraviolet transmission instrument or a gel imaging system. The quality of the RNA sample is evaluated according to the clarity and integrity of the 28S, 18S and 5S rRNA bands.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology experimental technology, specifically relating to a method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection. Background Technology

[0002] In molecular biology research, assessing the integrity of RNA extracted from samples (such as fungi, plants, and animal tissues) is a crucial quality control step for subsequent experiments such as reverse transcription, real-time quantitative PCR, and transcriptome sequencing. Agarose gel electrophoresis is the most commonly used method for detecting RNA integrity, determining whether RNA has degraded by observing the ratio of clarity to brightness of 28S and 18S ribosomal RNA bands. RNA molecules are highly susceptible to degradation by RNases, which are widely present in the environment. Traditional DNA electrophoresis buffers (such as TAE and TBE) may be contaminated with RNases during preparation and storage. More importantly, during electrophoresis, the current generates Joule heat, causing the temperature of the buffer and gel in the electrophoresis tank to rise. This temperature increase not only exacerbates the activity of any RNases that may be present but may also directly cause thermal denaturation or hydrolysis of RNA molecules, resulting in tailing, blurred bands, or even disappearance of the electrophoretic pattern, failing to accurately reflect the original integrity of the RNA extract and leading to misjudgment.

[0003] Existing systems using dedicated denatured or non-denatured RNA electrophoresis buffers, with the following main ideas and steps: The first step is to prepare the specific buffer: Prepare buffers such as MOPS (3-(N-morpholino)propanesulfonic acid), formaldehyde gel electrophoresis buffer, or other commercially available RNA-specific buffers. These buffers usually require separate stock solutions or the purchase of ready-made kits; The second step is to prepare the gel: Agarose gel is prepared using a special buffer solution, and denaturing agents such as formaldehyde may be added to the gel to inhibit RNA secondary structure and RNase. The third step is the electrophoresis process: a special buffer solution is poured into the electrophoresis tank. Electrophoresis is usually carried out in a low-temperature environment (such as an ice bath) or a cold room (4°C) to inhibit RNA degradation caused by heat production and changes in the pH of the buffer solution.

[0004] Existing techniques can effectively protect RNA and obtain clear electrophoretic bands, but they have the following drawbacks: 1. High cost: Specialized reagents such as MOPS or commercial kits are significantly more expensive than conventional TAE / TBE; 2. The operation is cumbersome and toxic: Formaldehyde is volatile and carcinogenic, and the operation needs to be carried out in a fume hood, which is harmful to the health of laboratory personnel; the preparation of special buffer solutions involves many steps; 3. High equipment requirements: It requires a cold room or a large amount of ice water bath to maintain the low temperature of the entire electrophoresis system, which places certain requirements on laboratory conditions and is not convenient for routine rapid detection.

[0005] Therefore, there is an urgent need for a simple electrophoresis pretreatment method that does not rely on special chemical reagents, requires no additional cooling equipment, and can effectively protect RNA using only conventional buffer solutions. Summary of the Invention

[0006] Technical Problem Solved: Addressing the shortcomings of existing technologies, this invention provides a pre-cooling method for agarose gel electrophoresis buffer for RNA integrity detection. This method solves several problems encountered in conventional agarose gel electrophoresis using ordinary TAE or TBE buffers for RNA integrity detection. These problems include: RNA sample degradation due to buffer temperature rise during electrophoresis; reliance on expensive or toxic specialized chemical reagents, increasing experimental costs and health risks; complex operation procedures requiring specially prepared buffers; and inconvenience due to requirements for cooling equipment or environment during electrophoresis. This pre-cooling method is based on the principle of "cold accumulation-heat buffering," essentially bringing thermodynamic regulation forward to the electrophoresis preparation stage to passively counteract the heat generated during electrophoresis, thereby maintaining a low-temperature steady state of the system without active temperature control.

[0007] Purpose of the Invention: This application discloses a method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection. It provides a method that effectively prevents RNA degradation during electrophoresis using only the most common and inexpensive laboratory TAE or TBE buffers. This simplifies the pre-electrophoresis preparation process for RNA integrity detection, eliminating the need for special buffer preparation. It eliminates the dependence on a consistently low-temperature electrophoresis environment, allowing experiments to be conveniently performed in a room-temperature laboratory. This avoids the use of expensive, specially prepared, or potentially toxic dedicated RNA electrophoresis buffers (such as MOPS buffer systems), reducing experimental costs and simplifying the operation process. It is a pretreatment method for RNA agarose gel electrophoresis based on a physical-thermodynamic regulation strategy, particularly suitable for rapid, low-cost, and highly reliable detection of RNA integrity using conventional TAE or TBE buffers.

[0008] To achieve the above objectives, this application provides the following technical solution: A method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection specifically includes the following steps: Step 1, Buffer Preparation: Place TAE buffer and / or TBE buffer into a clean, sealed container; The second step is deep pre-cooling: place the sealed container containing AE buffer and / or TBE buffer in an ice bath environment for pre-cooling for ≥3 hours. Step 3, Gel Preparation and Sample Loading: Prepare an agarose gel with a mass-to-volume percentage (W / V) of 1.0%-2.0% using pre-cooled TAE buffer and / or TBE buffer. After the gel solidifies, place it in an electrophoresis tank and add pre-cooled TAE buffer and / or TBE buffer dropwise until the liquid level is 1-5 mm above the gel surface. The ratio of the total volume of the TAE buffer and / or TBE buffer to the volume of the agarose gel should be 3:1 to 10:1. Mix 2 μL of RNA sample with 2×RNA loading buffer, which consists of 95% (v / v) formamide, 0.02% (w / v) SDS, 0.02% (w / v) bromophenol blue, 0.02% (w / v) xylenecyanine FF, and 1 mM EDTA. The volume ratio of 2×RNA loading buffer is 1:1. After mixing, the buffer is loaded into the sample well. Step 4, room temperature electrophoresis: Place the electrophoresis tank in an environment of 20-25℃, turn on the power, and perform electrophoresis at a voltage of 5-8 V / cm gel length; Step 5, Imaging Analysis: After electrophoresis, the gel is removed, nucleic acid staining is performed, and images are observed and acquired using a UV transilluminator or gel imaging system. The quality of the RNA sample is assessed based on the clarity and integrity of the 28S, 18S, and 5S rRNA bands.

[0009] Furthermore, in the first step, the TAE buffer and / or TBE buffer are either working solutions or 50-fold concentration stock solutions. When the TAE buffer and / or TBE buffer are 50-fold concentration stock solutions, they are first diluted to 1 times the working solution concentration.

[0010] Furthermore, the clean, sealed container in the first step is a glass bottle or a plastic reagent bottle.

[0011] Furthermore, in the second step, the sealed container containing AE buffer and / or TBE buffer is placed in an ice bath environment to ensure that the temperature of AE buffer and / or TBE buffer drops uniformly to close to 0°C, so that AE buffer and / or TBE buffer can accumulate sufficient cold energy and provide a stable low-temperature environment for the electrophoresis process.

[0012] Furthermore, in the second step, the ice bath environment can also be a low-temperature environment that can bring the initial temperature of the buffer solution to 0-4°C, wherein the ice blocks in the low-temperature environment are in a -20°C freezer.

[0013] Furthermore, the pre-cooling time in the second step is dynamically adjusted according to the volume of TAE buffer and / or TBE buffer and the ambient temperature. When the buffer volume is ≥500 mL or the ambient temperature is >25℃, the pre-cooling time is extended to 4-6 hours or overnight.

[0014] Furthermore, in the third step, the concentration of agarose gel is 1%-2% by mass / volume percentage.

[0015] Furthermore, in the third step, before gel electrophoresis, the electrophoresis tank is pre-cooled at 4°C for 30 minutes to synergistically enhance the cooling effect.

[0016] Furthermore, in the fifth step, nucleic acid staining is performed using EB nucleic acid dye and / or GelRed nucleic acid dye.

[0017] Furthermore, in the fifth step, the RNA sample is total RNA derived from fungi, plants, animal tissues or cells. After electrophoresis, the clarity, brightness ratio and presence of tailing of the 28S, 18S and 5S rRNA bands are analyzed by nucleic acid fluorescence staining and imaging to assess RNA integrity.

[0018] Explanation of the principle: This invention does not rely on chemical modification or enzyme inhibitors to eliminate RNase or stabilize RNA structure. Instead, it regulates the thermal state of the reaction system through a physical temperature control strategy. Prolonged ice bath pre-cooling gives the buffer a low enthalpy. In the initial stage of electrophoresis, the Joule heat generated by the current is first used to raise the temperature of the buffer itself. Due to the low initial temperature and high heat capacity of the pre-cooled buffer, its temperature rise is significantly slowed, thus maintaining the entire electrophoresis system (buffer and gel) at a relatively low temperature throughout the electrophoresis cycle. Through thermodynamic regulation, the degradation problem caused by temperature rise during RNA electrophoresis is solved. Specifically, by sufficiently pre-cooling the conventional electrophoresis buffers TAE and / or TBE, sufficient "cold energy" is stored at the start of electrophoresis, effectively buffering the Joule heating effect during subsequent electrophoresis and maintaining the system temperature at a low level, thus effectively protecting the RNA. After treatment using this method, clear 28S, 18S, and 5S RNA can be observed during electrophoresis. rRNA bands; this condition can effectively inhibit the increase in RNase activity and the thermal hydrolysis reaction of RNA molecules that may be caused by temperature rise, thereby achieving the purpose of protecting RNA integrity. This method only uses conventional buffers and only requires one pre-cooling treatment before the experiment, without the need for continuous external cooling equipment, which greatly simplifies the experimental operation. The pre-cooling buffer has low initial enthalpy and high heat capacity. After electrophoresis starts, Joule heat is preferentially absorbed by the buffer and used for heating. Due to its low initial temperature, large mass and high thermal inertia, the system temperature rise rate is significantly reduced. Experiments have shown that under the premise of ≥3 hours of pre-cooling, the average temperature of the buffer and gel can be stably controlled in the range of 12-18℃ during the entire electrophoresis process (usually 30-60 minutes), which is far below the threshold of significant enhancement of RNase activity and the temperature of accelerated RNA thermal hydrolysis, thereby achieving dual thermal protection of RNA molecules.

[0019] This application provides a method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection, which has the following advantages compared with the prior art: 1. Excellent economic efficiency: The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection in this application can completely reuse the laboratory standard TAE / TBE buffer, reducing the cost per detection by more than 90%, and eliminating the need to purchase MOPS, formaldehyde or special kits. 2. Enhanced safety: The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection in this application completely eliminates the use of carcinogenic reagents such as formaldehyde, avoids forced operation in fume hoods, and significantly reduces occupational health risks; 3. Extremely simplified operation: The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection in this application replaces the original multi-step process of "pre-cooling buffer" in one step, which is "preparing special buffer + preparing ice bath + reserving cold room". Even beginners can complete it on the same day. 4. Highly reliable results: The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection proposed in this application has been verified by RNA from multiple sources such as Cryptococcus neoformans, Arabidopsis thaliana, and mouse liver tissue. The band resolution is comparable to that of the MOPS-cold room system, the 28S / 18S ratio is stable and there is no tailing, and the RIN values ​​are highly consistent. 5. Strong equipment compatibility: The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection proposed in this application is applicable to any brand of horizontal electrophoresis tank without modification or additional cooling modules, greatly expanding application scenarios such as mobile field laboratories, teaching classrooms, and rapid clinical testing. Attached Figure Description

[0020] Figure 1 The above image shows the verification results of the agarose gel electrophoresis buffer pre-cooling method used for RNA integrity detection in this application. The top image shows the electrophoresis buffer TAE, and the bottom image shows the electrophoresis buffer TBE. Both images show clear 28S, 18S, and 5S bands with no obvious signs of degradation, confirming that the present invention achieves the same level of protection. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this disclosure, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0022] TAE buffer: Tris-acetic acid-EDTA buffer, which is the nucleic acid electrophoresis buffer of this application; TBE buffer: Tris-boric acid-EDTA buffer, which is the nucleic acid electrophoresis buffer of this application with a resolution higher than TAE; RNase: ribonuclease, which is a class of enzymes in this application that can degrade RNA, exists in the environment and is very stable.

[0023] Example 1: A method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection, specifically including the following steps: Step 1, Buffer Preparation: Place TAE buffer and / or TBE buffer into a clean, sealed container; The second step is deep pre-cooling: place the sealed container containing AE buffer and / or TBE buffer in an ice bath environment for pre-cooling for ≥3 hours. Step 3, Gel Preparation and Sample Loading: Prepare an agarose gel with a mass-to-volume percentage (W / V) of 1.0%-2.0% using pre-cooled TAE buffer and / or TBE buffer. After the gel solidifies, place it in an electrophoresis tank and add pre-cooled TAE buffer and / or TBE buffer dropwise until the liquid level is 1-5 mm above the gel surface. The ratio of the total volume of the TAE buffer and / or TBE buffer to the volume of the agarose gel should be 3:1 to 10:1. Mix 2 μL of RNA sample with 2×RNA loading buffer, which consists of 95% (v / v) formamide, 0.02% (w / v) SDS, 0.02% (w / v) bromophenol blue, 0.02% (w / v) xylenecyanine FF, and 1 mM EDTA. The volume ratio of 2×RNA loading buffer is 1:1. After mixing, the buffer is loaded into the sample well. Step 4, room temperature electrophoresis: Place the electrophoresis tank in an environment of 20-25℃, turn on the power, and perform electrophoresis at a voltage of 5-8 V / cm gel length; Step 5, Imaging Analysis: After electrophoresis, the gel is removed, nucleic acid staining is performed, and images are observed and acquired using a UV transilluminator or gel imaging system. The quality of the RNA sample is assessed based on the clarity and integrity of the 28S, 18S, and 5S rRNA bands.

[0024] In the first step, the TAE buffer and / or TBE buffer are either the working solution or a 50-fold concentration stock solution. If the TAE buffer and / or TBE buffer are a 50-fold concentration stock solution, they should first be diluted to 1 times the working solution concentration. The clean, sealed container in the first step is a glass bottle or a plastic reagent bottle.

[0025] In the second step, the sealed container containing AE buffer and / or TBE buffer is placed in an ice bath environment to ensure that the temperature of the AE buffer and / or TBE buffer drops uniformly to near 0°C, allowing the AE buffer and / or TBE buffer to accumulate sufficient cold energy and provide a stable low-temperature environment for the electrophoresis process. The ice bath environment in the second step can also be a low-temperature environment that allows the buffer to reach an initial temperature of 0-4°C, wherein the crushed ice in the low-temperature environment is a -20°C freezer. The pre-cooling time in the second step is dynamically adjusted according to the volume of TAE buffer and / or TBE buffer and the ambient temperature. When the buffer volume is ≥500 mL or the ambient temperature is >25°C, the pre-cooling time is extended accordingly to 4-6 hours or overnight.

[0026] In the third step, the agarose gel concentration is 1%-2% by mass / volume. Before gel electrophoresis, the electrophoresis tank is pre-cooled at 4°C for 30 minutes to enhance the cooling effect. In the fifth step, nucleic acid staining is performed using EB nucleic acid dye and / or GelRed nucleic acid dye.

[0027] With adequate pre-cooling of the buffer solution, RNA electrophoresis can be performed at room temperature without additional cooling equipment. In the fifth step, the RNA sample is total RNA derived from fungi, plants, animal tissues, or cells. After electrophoresis, the clarity, brightness ratio, and presence of tailing of the 28S, 18S, and 5S rRNA bands are analyzed by nucleic acid fluorescence staining and imaging to assess RNA integrity.

[0028] Example 2: A method for pretreatment of agarose gel electrophoresis buffer for RNA integrity detection, wherein the TAE and / or TBE buffer for RNA electrophoresis is pre-cooled and stored at 0°C for ≥3 hours; RNA agarose gel electrophoresis is performed using the pre-cooled buffer.

[0029] Cryptococcus neoformans RNA integrity detection (standard procedure): Buffer pre-cooling: Take 100mL of 1× TAE buffer and put it into a 50mL centrifuge tube. After sealing, completely bury the tube in a crushed ice bath and let it stand for 4 hours to pre-cool. Gel preparation: Take 80 mL of pre-cooled TAE, add 1.5 g of agarose, microwave to dissolve, cool slightly to about 60°C, pour into a gel casting plate, and let stand at room temperature to solidify; Electrophoresis preparation: Place the gel into the electrophoresis tank and inject the remaining 20 mL of pre-cooled TAE until it submerges the gel surface; Sample loading and electrophoresis: Mix 2 μL of Cryptococcus neoformans total RNA with 2 μL of 2× RNA loading buffer, and load the sample; cover the electrophoresis tank and electrophores at a constant voltage of 120V (≈6V / cm) for 45 minutes at room temperature of 22℃; Imaging analysis: EB staining for 15 minutes, destaining with deionized water, and image acquisition using a gel imaging system. Results showed that the 28S and 18S bands were sharp and well-separated, with a brightness ratio of 1.9:1, visible at 5S, and without tailing, indicating excellent RNA integrity.

[0030] Example 3, Adaptability verification under different pre-cooling conditions: The following tests were conducted: 1. Pre-cooling at -20℃ for 3 hours; 2. Pre-cooling in crushed ice (≈-5℃) for 4 hours; 3. Pre-cooling a large volume (500mL) of TBE in an ice bath at 25℃ for 6 hours. Clear rRNA bands were obtained in all tests, confirming that the pre-cooling medium and time had reasonable adjustable margins, and that the buffer solution did not freeze.

[0031] Example 4, Enhanced effect of synergistic pre-cooling in electrophoresis tank: An empty electrophoresis tank was placed in an ice bath 30 minutes in advance, and then pre-cooling buffer was injected. The average temperature throughout the electrophoresis process was measured to be about 2.3°C lower than that of the unpre-cooled tank, and the band sharpness was slightly improved, further confirming the feasibility of synergistic cooling.

[0032] The above description is only a preferred embodiment of the present invention. It should be noted that, under the premise of following the principles of the present invention, improved experimental schemes should also be considered within the scope of protection of the present invention.

Claims

1. A method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection, characterized in that, Specifically, the steps include the following: Step 1, Buffer Preparation: Place TAE buffer and / or TBE buffer into a clean, sealed container; The second step is deep pre-cooling: place the sealed container containing AE buffer and / or TBE buffer in an ice bath environment for pre-cooling for ≥3 hours. Step 3, Gel Preparation and Sample Loading: Prepare an agarose gel with a mass percentage concentration of 1.0%-2.0% using pre-cooled TAE buffer and / or TBE buffer. After the gel solidifies, place it in an electrophoresis tank and add pre-cooled TAE buffer and / or TBE buffer dropwise until the liquid level is 1-5 mm above the gel surface. The ratio of the total volume of the TAE buffer and / or TBE buffer to the volume of the agarose gel should be 3:1 to 10:

1. Mix 2 μL of RNA sample with 2×RNA loading buffer, which consists of 95% (v / v) formamide, 0.02% (w / v) SDS, 0.02% (w / v) bromophenol blue, 0.02% (w / v) xylenecyanine FF, and 1 mM EDTA. The volume ratio of 2×RNA loading buffer is 1:

1. After mixing, the buffer is loaded into the sample well. Step 4, room temperature electrophoresis: Place the electrophoresis tank in an environment of 20-25℃, turn on the power, and perform electrophoresis at a voltage of 5-8 V / cm gel length; Step 5, Imaging Analysis: After electrophoresis, the gel is removed, nucleic acid staining is performed, and images are observed and acquired using a UV transilluminator or gel imaging system. The quality of the RNA sample is assessed based on the clarity and integrity of the 28S, 18S, and 5S rRNA bands.

2. The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection according to claim 1, characterized in that, In the first step, the TAE buffer and / or TBE buffer are either the working solution or a 50-fold concentration of the storage solution. When the TAE buffer and / or TBE buffer are a 50-fold concentration of the storage solution, they should be diluted to 1 times the working solution concentration first.

3. The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection according to claim 1, characterized in that, The clean, sealed container used in the first step is a glass bottle or a plastic reagent bottle.

4. The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection according to claim 1, characterized in that, In the second step, the sealed container containing AE buffer and / or TBE buffer is placed in an ice bath environment to ensure that the temperature of AE buffer and / or TBE buffer drops uniformly to close to 0°C, so that AE buffer and / or TBE buffer can accumulate sufficient cold energy and provide a stable low temperature environment for the electrophoresis process.

5. The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection according to claim 4, characterized in that, In the second step, the ice bath environment can also be a low-temperature environment that can bring the initial temperature of the buffer solution to 0-4°C, such as a -20°C refrigerator or crushed ice.

6. The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection according to claim 1, characterized in that, In the second step, the pre-cooling time is dynamically adjusted according to the volume of TAE buffer and / or TBE buffer and the ambient temperature. When the buffer volume is ≥500 mL or the ambient temperature is >25℃, the pre-cooling time is extended to 4-6 hours or overnight.

7. The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection according to claim 1, characterized in that, In the third step, the concentration of agarose gel is 1%-2% by mass / volume percentage.

8. The agarose gel electrophoresis buffer pre-cooling method for RNA integrity detection according to claim 1, characterized in that, In the third step, before gel electrophoresis, the electrophoresis tank is pre-cooled at 4°C for 30 minutes to enhance the cooling effect.

9. The method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection according to claim 1, characterized in that, In the fifth step, nucleic acid staining is performed using EB nucleic acid dye and / or GelRed nucleic acid dye.

10. The method for pre-cooling agarose gel electrophoresis buffer for RNA integrity detection according to claim 1, characterized in that, In the fifth step, the RNA sample is total RNA derived from fungi, plants, animal tissues or cells. After electrophoresis, the clarity, brightness ratio and presence of tailing of the 28S, 18S and 5S rRNA bands are analyzed by nucleic acid fluorescence staining and imaging to assess RNA integrity.

Images