制样方法对菊花花蕾激光显微切割样品RNA提取质量的影响

Effect of Sample Preparation Methods on the Quality of RNA Extraction from Chrysanthemum Bud Laser Microdissection Samples

[目的]研究不同固定剂及切片制样方式对菊花花蕾激光显微切割样品RNA提取质量的影响。[方法]以丙酮和乙醇冰醋酸2种试剂固定菊花花蕾,采用石蜡和冰冻切片2种方式制作样本,显微切割并收集最外两轮小花原基,提取RNA后比较提取质量。[结果]石蜡和经梯度蔗糖保护的冰冻切片均能很好地保存花蕾组织形态,就石蜡切片而言,乙醇冰醋酸固定组织切割样品的RNA降解明显低于丙酮固定样品;而2种固定剂的冰冻切片切割样品RNA提取质量无明显差异。与石蜡切片相比,冰冻切片显微切割样品RNA质量更高。切割时收集管盖加入RNA提取缓冲液可明显缓解RNA降解。显微切割的不同品种菊花花原基与花发育有关的基因表达水平存在差异,表明切割获得的材料可用于下一步研究。[结论]植物组织经乙醇冰醋酸固定、梯度蔗糖保护后冰冻切片进行显微切割效果较佳,可富集特异组织或细胞并从中获得较高质量的RNA。

[Objective]To study the effect of different fixatives and section methods on RNA quality of flower primordia of Chrysanthemum buds collected by microdissection.[Method]Chrysanthemum buds was fixed by acetone or ethanol acetic acid and then paraffin section/frozen section was prepared for laser microdissection respectively.Outermost two whorls of flower primordia were identified and collected by laser microdissection.The quantity and quality of isolated RNA were assessed.[Result]Both tissues embedded in paraffin for paraffin section or OCT for cryosection preserves well the anatomical structure and morphology of the buds.For paraffin-section,ethanol acetic acid was a better choice being a fixative than that of acetone,as that RNA extracted from the microdissection samples was less degraded.There was no significant difference in RNA integrity of cryosection between these two fixatives.The tissues conducted cryosection could get higher quality of RNA compared with that of paraffin-section.Furthermore,when microdissection was conducted,containing the extraction buffer from RNA isolation kit in the cap of PCR tube could preserved RNA integrity of the samples.Genes expression of flowering locus T in flower primordia was different in species of chrysanthemum showed that samples captured by microdissection can be used for subsequent research.[Conclusion]Plant tissues fixed by ethanol-acetic acid and protected by gradient sucrose before cryosection could collect specific cell-type populations by laser microdissection.From those populations we could obtain enough RNA with high integrity.