Establishment of trace RNA extraction from pistils by the Laser Capture Microdissection in Japanese apricot

Abstract:【Objective】The purpose of the experiment is to establish a technology system that is suitable for the extraction of trace RNA by laser capture microdissection in Japanese apricot. The paraffin sec- tion was used as a carrier, and the sample material was separated with the help of the laser capture mi- crodissection system, and then the cut tissue was used to extract trace RNA. The aim was to solve the problems that the pistil was too small to separate the tissues and the trace RNA was not easy to extract by general extraction method.【Methods】Using the flower buds and pistils of Japanese apricot varieties‘Daqiandi’and‘Longyan’as experimental materials, the paraffin sections were made. The strips with different thicknesses of 6, 8, 10, 12 and 14 μm were set, and then the cutting effects of strips were com- pared with different thicknesses and the best strip thickness was selected to make slices. The Leica LMD 7000 laser capture microdissection system was used as the core of the experiment. Different laser intensities of 20, 30 and 40 were set up under the premise of determining the best strip thickness, and the cutting effect of different laser intensities on the slice was compared, the laser intensity with the best cutting effect was selected to cut the pistils, and then the quality of the RNA extracted from the material  was compared after either normal temperature or liquid nitrogen preservation. Finally, an optimal sys- tem for extracting trace RNA from paraffin sections and laser capture microdissection was established.【Results】Two varieties of full flower buds were selected to make paraffin sections. In the process of paraffin section production, the tested materials should be stored in a 4 °C refrigerator, except for the wax infiltration and slices making processes. Other steps, such as dehydration, transparency and so on were operated in the 4 °C refrigerator, before making slices, The section method of this experiment was longitudinal cutting. After repeated comparisons, the 10 μm, 12 μm, and 14 μm strips can be attached to the PEN membrane completely, which were not easy to break, and there was no gap between the strip and the membrane; However, 6 μm and 8 μm strips were easy to break when they were contacted with water, and they were not easy to stick; Slices with a thickness of 14 μm cannot be cut directly at one time; When the thickness of the slice was 12 μm, sometimes it can be cut off at one time, but sometimes the reverse was true; When the thickness of the slice was 10 μm, it can be cut off at one time; The opti- mal slice thickness of paraffin sections was determined to be 10 μm in this experiment. When the laser intensity was set to 20, it was not enough to cut off the material at one time; when the laser intensity was set to 30, the material can be cut off at one time, and the cutting edge had less loss to the tissue; When the laser intensity was set to 40, the material can be cut off at one time, but the cutting edge was more burnt than the laser intensity set at 30, which affected the integrity of the tissue; After comparison, the laser intensity was finally set to 30 in this experiment. The RIN value of the RNA extracted from the cutting material stored in liquid nitrogen was 4.1, and the RIN value of the RNA extracted from the cut- ting material stored at room temperature was 2.4, and the concentration of the former was much higher than that of the latter. And it was clear that the RIN value of the RNA extracted from the cutting materi- al stored in liquid nitrogen was higher than that stored at room temperature.【Conclusion】This experi- ment improved the storage temperature of materials in certain steps during the paraffin section making process, and determined the slice thickness suitable for Japanese apricot buds and the laser intensity for laser capture microdissection. Therefore, a relatively simple, stable and reproducible system using paraf- fin sections with the laser capture microdissection to extract trace RNA was established, which could provide a basis for the study on trace RNA extraction and subsequent molecular experiments of woody plants like Japanese apricot. However, the experimental results of paraffin sections were not as good as frozen sections. Paraffin sections manufacture was more complicated and required longer cycles, and could not avoid the negative effects of organic solvents on RNA, so it should be studied further how to streamline the paraffin section production process and production cycle, to reduce the negative impact of organic solvents on RNA, and to apply the frozen section technology to plants in a complete manner without adversely affecting the plant materials, like the effect of frozen section on plant big vacuole.