Plant tissue paraffin embedding method of high-fidelity RNA (Ribonucleic Acid)

Abstract

The invention relates to the technical field of plant microscopic observation, in particular to a plant tissue paraffin embedding method of high-fidelity RNA (Ribonucleic Acid). The invention provides a paraffin embedding method for plant tissue of high-fidelity RNA (Ribonucleic Acid), which comprises the following steps: firstly mixing the tissue and a stationary liquid under negative pressure, and then replacing the stationary liquid for fixing, carrying out ethanol solution gradient dehydration on the fixed tissue; the dehydrated tissue is firstly placed in a transparent agent to be treated, and then xylene is used for treatment; mixing the transparent tissue and the mixed wax solution at 35-39 DEG C, heating to 58-62 DEG C, mixing to obtain a mixture, mixing the mixture with the paraffin solution, and embedding to obtain an embedded sample. According to the method, RNA degradation in the paraffin embedding process is reduced, it is ensured that RNA extracted from an embedded sample has high quality, operation is easy, complex equipment is not needed, and the experiment time is short.

Description

Technical Field

[0001] The invention relates to the technical field of plant microscopic observation, and in particular to a plant tissue paraffin embedding method for high-fidelity RNA. Background Art

[0002] Paraffin embedding technology is widely used in section staining, in situ hybridization and microdissection of animal and plant tissue samples. However, how to prevent RNA degradation during paraffin embedding has always been a technical difficulty, especially for molecular biology research and RNA sequencing that require high-quality RNA extraction. Traditional embedding steps usually cannot effectively retain RNA in tissues, resulting in low quality of subsequently extracted RNA, which cannot meet the requirements of downstream applications such as high-throughput sequencing.

[0003] Although laser microdissection technology provides an efficient means for precise cutting of paraffin-embedded samples, it only solves the problem of cutting accuracy and cannot solve the problem of RNA loss. The reasons for the poor RNA integrity can be traced back to many deficiencies in the paraffin section production process, including tissue fixation, wax immersion, embedding and other steps, among which the fixative and temperature have a greater impact on the quality of RNA.

[0004] Based on this, there is an urgent need to provide an improved solution that can preserve RNA integrity with high quality during paraffin embedding. Summary of the invention

[0005] The object of the present invention is to provide a plant tissue paraffin embedding method which can reduce RNA degradation during paraffin embedding and ensure that RNA extracted from embedded samples has high quality.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for paraffin embedding of plant tissues for high-fidelity RNA, comprising the following steps: (1) Mix the tissue and fixative at a pressure of -0.6 to -1.0 KPa for 10 to 20 minutes, replace the fixative to fix the tissue, and obtain the fixed tissue; (2) placing the fixed tissue in a gradient ethanol solution with a mass fraction of 70%, 80%, 90%, and 100% in sequence for dehydration to obtain dehydrated tissue; (3) Place the dehydrated tissue in a clearing agent for 1.8 to 2.2 hours and then treat with xylene to obtain a clear tissue; (4) Mix the transparent tissue and the mixed wax solution at 35-39°C for 16-20 hours, heat to 58-62°C and mix for 0.8-1.2 hours to obtain a mixture; mix the mixture with the paraffin solution for 3-5 hours to obtain the wax-impregnated tissue; (5) Embed the wax-soaked tissue to obtain an embedded sample.

[0007] Preferably, in step (1), the fixing solution comprises anhydrous ethanol and glacial acetic acid; The volume ratio of the anhydrous ethanol to the glacial acetic acid is 3:1.

[0008] Preferably, in step (1), the volume ratio of the tissue and the fixative is 1:50-70.

[0009] Preferably, in step (1), the fixing is rotation fixing, the rotation speed of the rotation fixing is 6-10 rpm; the fixing time is 1.8-2.2 h; and the fixing temperature is 3-5°C.

[0010] Preferably, in step (2), the time of each dehydration is 30-50 minutes; the dehydration is rotary dehydration, the rotation speed of the rotary dehydration is 6-10 rpm; the dehydration temperature is 3-5°C.

[0011] Preferably, in step (3), the transparent agent comprises anhydrous ethanol and xylene; the volume ratio of the anhydrous ethanol to xylene is 1:0.5-1.5.

[0012] Preferably, in step (3), the number of times of treatment with xylene is 2 to 3 times.

[0013] Preferably, in step (4), the mixed wax liquid comprises xylene and liquid paraffin; the volume ratio of xylene to liquid paraffin is 1-3:2.

[0014] Preferably, in step (5), the embedding temperature is 60-62°C.

[0015] The present invention also provides the application of the embedded sample obtained by the plant tissue paraffin embedding method in molecular biological analysis.

[0016] Beneficial effects of the present invention: The invention provides a paraffin embedding method for plant tissues of high-fidelity RNA. The method pre-treats the tissues under a certain pressure with a fixative before fixation, thereby reducing RNA degradation in the sample. Then, the fixative is replaced and the tissue is rotated and fixed at a low temperature, thereby further alleviating RNA degradation and obtaining a fixed tissue. The fixed tissue is dehydrated at a low temperature, and then transparentized, wax-impregnated and embedded to obtain an embedded sample. The embedded sample is cut by laser microdissection, and the obtained cell and tissue sections are extracted by an RNA kit. The obtained RNA is tested for integrity RIN value, and the RIN value is stabilized at 6 or above, thereby significantly improving the RNA preservation effect and solving the technical problem of RNA being easily degraded in the traditional method.

[0017] The plant tissue paraffin embedding method of the present invention greatly reduces RNA degradation during the paraffin embedding process, ensures that the RNA extracted from the embedded sample has high quality, and is suitable for downstream molecular biology research and molecular biology analysis such as RNA sequencing analysis, which is of great significance for samples requiring RNA sequencing or expression analysis; and is simple to operate, does not require complex equipment, and has a short experimental time. It is suitable for paraffin embedding of various plant tissues, and can effectively retain RNA integrity regardless of the size and complexity of the tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The laser microscopic section is of an embedded rice mature embryo sac sample obtained by the paraffin embedding method of plant tissue described in Example 1; Figure 2 The RNA quality test result of the embedded rice ovule sample obtained by the plant tissue paraffin embedding method described in Example 1; Figure 3 The RNA quality test results of the embedded rice ovule samples obtained by the traditional plant tissue paraffin embedding method described in Comparative Example 1; Figure 4 It is a laser microscopic section of a mature embryo sac sample of embedded water lily obtained by the paraffin embedding method of plant tissue described in Example 1; Figure 5 This is the RNA quality test result of the embedded water lily ovule sample obtained by the plant tissue paraffin embedding method described in Example 1. DETAILED DESCRIPTION

[0019] A method for paraffin embedding of plant tissues for high-fidelity RNA comprises the following steps: (1) Mix the tissue and fixative at a pressure of -0.6 to -1.0 KPa for 10 to 20 minutes, replace the fixative to fix the tissue, and obtain the fixed tissue; (2) placing the fixed tissue in a gradient ethanol solution with a mass fraction of 70%, 80%, 90%, and 100% in sequence for dehydration to obtain dehydrated tissue; (3) Place the dehydrated tissue in a clearing agent for 1.8 to 2.2 hours and then treat with xylene to obtain a clear tissue; (4) Mix the transparent tissue and the mixed wax solution at 35-39°C for 16-20 hours, heat to 58-62°C and mix for 0.8-1.2 hours to obtain a mixture; mix the mixture with the paraffin solution for 3-5 hours to obtain the wax-impregnated tissue; (5) Embed the wax-soaked tissue to obtain an embedded sample.

[0020] In the present invention, in step (1), since a large amount of tissue is extracted during the experiment and the extraction time is long, in order to ensure the morphology and structure of the tissue, the tissue needs to be placed in a fixative for short-term preservation, and the fixative is replaced every 10 minutes. After all tissues are extracted, the fixative is replaced and the treatment is performed under a certain pressure.

[0021] In the present invention, in step (1), the fixing solution comprises anhydrous ethanol and glacial acetic acid; The volume ratio of anhydrous ethanol to glacial acetic acid is 3:1; The pressure is preferably -0.7~0.9KPa, more preferably -0.8KPa; The pressure regulation method is vacuuming; The mixing time is preferably 13 to 17 minutes, more preferably 15 minutes; The stationary phase needs to be precooled at 3-5°C, preferably 4°C, before mixing.

[0022] In the present invention, in step (1), the volume ratio of the tissue and the fixative is 1:50-70, preferably 1:55-65, and more preferably 1:60; The tissue and fixative need to be mixed on ice; In the present invention, in step (1), the fixing is rotation fixing, and the rotation speed of the rotation fixing is 6-10 rpm, preferably 7-9 rpm, and more preferably 8 rpm; The fixed time is 1.8 to 2.2 hours, preferably 1.9 to 2.1 hours, and more preferably 2 hours; The fixed temperature is 3-5°C, preferably 4°C.

[0023] In the present invention, in step (2), the dehydration time of each gradient ethanol solution is 30 to 50 minutes, preferably 35 to 45 minutes, and more preferably 40 minutes; The dehydration is a rotary dehydration, and the rotation speed of the rotary dehydration is 6 to 10 rpm, preferably 7 to 9 rpm, and more preferably 8 rpm; The dehydration temperature is 3-5°C, preferably 4°C.

[0024] In the present invention, in step (3), the transparent agent comprises anhydrous ethanol and xylene; the volume ratio of the anhydrous ethanol to xylene is 1:0.5-1.5, preferably 1:0.8-1.2, and more preferably 1:1; The treatment time is preferably 1.9 to 2.1 hours, more preferably 2 hours.

[0025] In the present invention, in step (3), the number of times of treatment with xylene is 3 times, the time of the first treatment is 1-3 s, preferably 2 s; the time of the second treatment is 35-45 min, preferably 38-42 min, more preferably 40 min; the time of the third treatment is 25-35 min, preferably 28-32 min, more preferably 30 min.

[0026] In the present invention, in step (4), the mixed wax liquid comprises xylene and liquid paraffin; the volume ratio of xylene to liquid paraffin is 1 to 3:2, preferably 2:2; The temperature at which the transparent tissue and the mixed wax liquid are mixed is preferably 36-38° C., and more preferably 37° C.; the time for mixing the transparent tissue and the mixed wax liquid is preferably 17-19 hours, and preferably 18 hours; The temperature after heating is preferably 59-61°C, more preferably 60°C; The mixing time after heating is preferably 0.9 to 1.1 hours, more preferably 1 hour; The mixing time of the mixture and the paraffin solution is preferably 3.5 to 4.5 hours, more preferably 4 hours; During the mixing of the mixture and the paraffin solution, the paraffin solution is replaced every 0.8 to 1.2 hours, preferably every 0.9 to 1.1 hours, and more preferably every 1 hour.

[0027] In the present invention, in step (5), the embedding temperature is 60-62°C, preferably 61°C.

[0028] The present invention also provides the application of the embedded sample obtained by the plant tissue paraffin embedding method in molecular biological analysis.

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1 A method for high-fidelity RNA paraffin embedding in plant tissue (1) Mix the tissue and fixative (anhydrous ethanol and glacial acetic acid mixed in a volume ratio of 3:1 and precooled at 4°C) in a volume ratio of 1:50 on ice for 15 minutes. During the mixing process, vacuum is required to maintain the pressure at -0.8KPa. Then replace the fixative and rotate at 4°C and 8rpm for 2 hours to obtain the fixed tissue. (2) The fixed tissue was placed in gradient ethanol solutions with mass fractions of 70%, 80%, 90%, and 100% in sequence, and spin-dehydrated at 4°C and 8 rpm. The dehydration time for each gradient ethanol solution was 30 min to obtain the dehydrated tissue; (3) The dehydrated tissue was placed in a transparent agent (anhydrous ethanol and xylene mixed in a volume ratio of 1:1) for 2 h, and then treated with xylene for 1 s, 40 min, and 30 min in sequence to obtain a transparent tissue; (4) Mix the transparent tissue and the mixed wax solution (mix xylene and liquid paraffin in a volume ratio of 3:2) in a 37°C incubator for 16 hours, and then place it in a 60°C hybridization oven for 1 hour to obtain a mixture; mix the mixture with the paraffin solution for 4 hours, replacing the paraffin solution every 1 hour, to obtain the wax-impregnated tissue; (5) Embed the wax-soaked tissue at 62°C to obtain an embedded sample.

[0031] Example 2 A method for high-fidelity RNA paraffin embedding in plant tissue (1) Mix the tissue and fixative (anhydrous ethanol and glacial acetic acid mixed in a volume ratio of 3:1 and precooled at 4°C) in a volume ratio of 1:60 on ice for 20 minutes. During the mixing process, vacuum is required to maintain the pressure at -1.0 KPa. Then replace the fixative and rotate at 5°C and 6 rpm for 1.8 hours to obtain the fixed tissue. (2) The fixed tissue was placed in gradient ethanol solutions with mass fractions of 70%, 80%, 90%, and 100% in sequence, and spin-dehydrated at 5°C and 6 rpm. The dehydration time for each gradient ethanol solution was 40 min to obtain the dehydrated tissue; (3) The dehydrated tissue was placed in a transparent agent (anhydrous ethanol and xylene mixed in a volume ratio of 1:1.5) for 1.8 h, and then treated with xylene for 3 s, 45 min, and 35 min in sequence to obtain a transparent tissue; (4) Mix the transparent tissue and the mixed wax solution (mix xylene and liquid paraffin in a volume ratio of 2:2) in a 38°C incubator for 18 hours, and then place it in a 58°C hybridization oven for 1.2 hours to obtain a mixture; mix the mixture with the paraffin solution for 4.5 hours, replacing the paraffin solution every 1.2 hours to obtain the wax-impregnated tissue; (5) Embed the wax-soaked tissue at 60°C to obtain an embedded sample.

[0032] Example 3 A method for paraffin embedding of plant tissues for high-fidelity RNA (1) Mix the tissue and fixative (anhydrous ethanol and glacial acetic acid mixed in a volume ratio of 3:1 and precooled at 4°C) in a volume ratio of 1:70 on ice for 18 minutes. During the mixing process, vacuum is required to maintain the pressure at -0.6 KPa. Then replace the fixative and rotate at 3°C ​​and 10 rpm for 2.2 hours to obtain the fixed tissue. (2) The fixed tissue was placed in gradient ethanol solutions with mass fractions of 70%, 80%, 90%, and 100% in sequence, and spin-dehydrated at 3°C ​​and 10 rpm. The dehydration time of each gradient ethanol solution was 50 min to obtain the dehydrated tissue; (3) The dehydrated tissue was placed in a transparent agent (anhydrous ethanol and xylene mixed in a volume ratio of 1:0.5) for 2.2 h, and then treated with xylene for 2 s, 35 min, and 25 min in sequence to obtain a transparent tissue; (4) Mix the transparent tissue and the mixed wax solution (mix xylene and liquid paraffin in a volume ratio of 1:2) in a 36°C incubator for 20 hours, and then place in a 62°C hybridization oven for 0.8 hours to obtain a mixture; mix the mixture with the paraffin solution for 3.5 hours, replacing the paraffin solution every 0.8 hours to obtain the wax-impregnated tissue; (5) Embed the wax-soaked tissue at 61°C to obtain an embedded sample.

[0033] Comparative Example 1 Traditional plant tissue paraffin embedding method (1) Mix the tissue and fixative (anhydrous ethanol and glacial acetic acid mixed in a volume ratio of 1:1 and precooled at 4°) in a volume ratio of 1:50 for 2 hours to obtain fixed tissue; (2) The fixed tissue was placed in gradient ethanol solutions with mass fractions of 50%, 70%, 85%, 95%, 100%, and 100% in sequence, and dehydrated at 4°C. The dehydration time in each gradient ethanol solution was 5 hours, and then dehydrated in 100% ethanol solution at room temperature for 2 hours to obtain the dehydrated tissue; (3) Place the dehydrated tissue in a transparent agent (anhydrous ethanol and xylene mixed in a volume ratio of 1:1) for 1 hour, and then treat it with xylene three times, each treatment time is 1 hour, to obtain a transparent tissue; (4) Mix the transparent tissue and the mixed wax solution (mix xylene and liquid paraffin in a volume ratio of 1:1) at 58°C for 16 hours, and mix the mixture with the paraffin solution at 58°C for 72 hours. During this period, the paraffin solution is replaced every 24 hours to obtain the wax-impregnated tissue; (5) Embed the wax-soaked tissue at 62°C to obtain an embedded sample.

[0034] Experimental Example 1

[0035] Fresh rice ovules were taken, and the stigma and placenta were removed. During the extraction process, since a large amount of tissues were extracted during the experiment and the extraction time was long, in order to ensure the morphology and structure of the tissues, the tissues needed to be placed in a fixative for short-term preservation, and the fixative was replaced every 10 minutes. After all tissues were extracted, the fixative was replaced, and the fresh rice ovules were embedded using the methods described in Example 1 and Comparative Example 1 to obtain embedded samples; The embedded samples were sliced ​​to a thickness of 10 μm, and the 10 μm slices were placed on glass slides, developed at 35°C, dewaxed with xylene for 10 min, dried, and then laser microsectioned to obtain tissue sections (such as Figure 1 As shown in the figure), RNA was extracted using the PicoPure™ RNA Isolation Kit (kit0214). The quality of the extracted RNA was tested by capillary electrophoresis using an Agilent Bioanalyzer and evaluated using the RIN (RNA Integrity Number) score of the software, with 10 indicating the best RNA integrity and 0 indicating the worst. The results are shown in the figure below. Figure 2~3 shown.

[0036] from Figure 2~3 It can be seen that the rice ovules were paraffin-embedded using the plant tissue paraffin embedding method described in Example 1, and RNA was extracted from the embedded samples. The extracted RNA had good integrity and was suitable for RNA sequencing, and the measured RIN score was 6.9; the rice ovules were paraffin-embedded using the traditional plant tissue paraffin embedding method described in Comparative Example 1, and RNA was extracted from the embedded samples, and the measured RIN score was 1.1; it can be seen that the plant tissue paraffin embedding method described in Example 1 significantly reduces RNA degradation compared with the traditional plant tissue paraffin embedding method described in Comparative Example 1.

[0037] Experimental Example 2 Fresh ovules of water lilies are taken, and attached tissues are removed. During the extraction process, since the amount of tissues extracted during the experiment is large and the extraction time is long, in order to ensure the morphology and structure of the tissues, the tissues need to be placed in a fixative for short-term preservation, and the fixative is replaced every 10 minutes. After all tissues are extracted, the fixative is replaced, and the fresh ovules of water lilies are embedded using the method described in Example 1 to obtain embedded samples; The embedded samples were sliced ​​to a thickness of 10 μm, and the 10 μm slices were placed on glass slides, developed at 35°C, dewaxed with xylene for 10 min, dried, and then laser microsectioned to obtain tissue sections (such as Figure 4As shown in the figure), RNA was extracted using the PicoPure™ RNA Isolation Kit (kit0214). The quality of the extracted RNA was tested by capillary electrophoresis using an Agilent Bioanalyzer and evaluated using the RIN (RNA Integrity Number) score of the software, with 10 indicating the best RNA integrity and 0 indicating the worst. The results are shown in the figure below. Figure 5 shown.

[0038] from Figure 5 It can be seen that the water lily ovules were paraffin-embedded using the plant tissue paraffin embedding method described in Example 1, and RNA was extracted from the embedded samples. The extracted RNA had good integrity and was suitable for RNA sequencing. The measured RIN score was 6.

[0039] As can be seen from the above examples, the present invention provides a method for paraffin embedding of plant tissues with high-fidelity RNA. The method reduces RNA degradation during paraffin embedding, ensures that RNA extracted from embedded samples has high quality, is simple to operate, does not require complex equipment, and has a short experimental time.

[0040] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for paraffin embedding of plant tissues for high-fidelity RNA, characterized in that: The steps include: (1) Mix the tissue and fixative at a pressure of -0.6 to -1.0 KPa for 10 to 20 minutes, replace the fixative to fix the tissue, and obtain the fixed tissue; (2) placing the fixed tissue in a gradient ethanol solution with a mass fraction of 70%, 80%, 90%, and 100% in sequence for dehydration to obtain dehydrated tissue; (3) Place the dehydrated tissue in a clearing agent for 1.8 to 2.2 hours and then treat with xylene to obtain a clear tissue; (4) Mix the transparent tissue and the mixed wax solution at 35-39°C for 16-20 hours, heat to 58-62°C and mix for 0.8-1.2 hours to obtain a mixture; mix the mixture with the paraffin solution for 3-5 hours to obtain the wax-impregnated tissue; (5) Embedding the wax-impregnated tissue to obtain an embedded sample; In step (1), the fixing solution includes anhydrous ethanol and glacial acetic acid.

2. The method for paraffin embedding of plant tissue according to claim 1, characterized in that: In step (1), the fixing solution comprises anhydrous ethanol and glacial acetic acid; The volume ratio of the anhydrous ethanol to the glacial acetic acid is 3:

1.

3. The method for paraffin embedding of plant tissue according to claim 2, characterized in that: In step (1), the volume ratio of the tissue and the fixative is 1:50-70.

4. The method for paraffin embedding of plant tissue according to claim 3, characterized in that: In step (1), the fixing is rotation fixing, the rotation speed of the rotation fixing is 6-10 rpm; the fixing time is 1.8-2.2 h; the fixing temperature is 3-5°C.

5. The method for paraffin embedding of plant tissue according to claim 4, characterized in that: In step (2), the time of each dehydration is 30-50 minutes; the dehydration is rotary dehydration, the rotation speed of the rotary dehydration is 6-10 rpm; the dehydration temperature is 3-5°C.

6. The method for paraffin embedding of plant tissue according to claim 5, characterized in that: In step (3), the transparent agent includes anhydrous ethanol and xylene; the volume ratio of the anhydrous ethanol to xylene is 1:0.5~1.

5.

7. The method for paraffin embedding of plant tissue according to claim 6, characterized in that: In step (3), the number of times of treatment with xylene is 2 to 3 times.

8. The method for paraffin embedding of plant tissue according to claim 7, characterized in that: In step (4), the mixed wax liquid includes xylene and liquid paraffin; the volume ratio of xylene to liquid paraffin is 1-3:

2.

9. The method for paraffin embedding of plant tissue according to claim 8, characterized in that: In step (5), the embedding temperature is 60-62°C.

10. Use of the embedded sample obtained by the plant tissue paraffin embedding method according to any one of claims 1 to 9 in molecular biological analysis.

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