The Laser Microdissection System Of Chinese Fir And Popuplus Cambium And RNA Quality Control

Laser microdissection (LMD) is a technique which can isolate specific tissues, cell types and even organelles from sectioned biological specimen under the microscope in a manner conducive to the extraction of RNA, DNA or protein. Pioneering research has demonstrated the potential of LMD as a powerful tool for investigating molecular and biochemical events that occur in discrete cells of complex, heterogeneous tissues, with the characteristic of fast, precise and easy-operation. LMD, which is an established technique in many areas of biology, has now been successfully adapted for use with plant tissues.Woody plants have the ability to produce secondary tissue (secondary xylem and phloem) from a meristematic tissue called vascular cambium, responsible for the radial growth of a tree. The research of expression and control of gene during the wood formation by molecular and cytological is an important scientific issue for scientists. However, the knowledge of cambium is limited, because it is hard to separate the cambium cells from other surrounding cells by traditional methods. Selecting the cambium cells precisely is a key point for the research of cambium cells. Since 1990s, the development of Laser Microdissection facilitated the collection of special materials.In the first step of research, we established a suitable cryo-sectioning method for stem organs of Chinese fir and Populus. With studying on the technical parameters of fixation and pretreatment of tissue-cultured material, a suitable procedure consisted of several steps is considered the best protocol as follows. The samples were first fixed in Canoy's fixation for 4 to 6 hours, and then saturated in 10% sucrose PBS solution for 6 to 12 hours. After embedded they were frozen in liquid nitrogen for 50 to 90 seconds and cryo-sectioned. The procedure of dehydration after cryo-sectioning improved the quality of section to a certain extent. The established protocol applied to wild Chinese fir and Populus successfully. The thinnest section is up to 10μm. The section is clear and the cell structure is easy to recognize. In this study, the cryosection of woody plant stem was systematically researched and made a solid foundation for the application of Laser Microdissection in woody plant's cell and molecular biology research.We used a Leica AS Laser Microdissection system for isolation of cells from prepared 12μm -thick stem cryosections of wild Chinese fir and Populus. Dissection conditions were optimized to obtain a clean, narrow excision of the selected cells: 40×XT objective at aperture 10～13, intensity 45～46, and speed 3～5. After about 6000 cambium cells were collected, we use Qiagen RNeasy Micro Kit to extract RNA. Because the sample yield that can be obtained by this method is too low for quality control of RNA using a standard technique such as ultraviolet spectrophotometer or agarose gel electrophoresis, RNA was demonstrated by RT-PCR firstly. RT-PCR was performed usingβ-Actin gene-specific primers to amplify a full-lengthβ-Actin gene. A fragment of about 400bp in length was detected by electrophoresis in agarose gels. Full-length Expansin 2 gene-specific primers was also used to amplify the cDNA which was come from cambium cells of Chinese fir, and a fragment of about 1400bp was detected. All of these experiments demonstrated the quality of RNA is fine.The RNA which was extracted from Populus 6000 cambium cells, was analyzed by Agilent 2100 Bioanalyzer. The results were: RNA concentration: 1,395pg/μl, rRNA Ratio [28S/18S]: 1.3;RNA Integrity Number [RIN]: 7.1. The data above proved that the RNA is of high quality without degradation. The sharp and high fluorescence peak suggested the purity of RNA without DNA determination. RNA yield factor was 2.325pg total RNA/cell. The quality of RNA reached the standards of cDNA microarray analysis, while it required linear amplification before applied to microarray analysis.This research applied the Laser Microdissection to woody plant for the first time, and made a technical foundation for the detection of important genes related to wood formation and wood character. This experimental system can also be applied to the research of development and genetics of other plants and organs.