制樣方法對菊花花蕾激光顯微切割樣品RNA提取質量的影響

摘要 ［目的］研究不同固定劑及切片制樣方式對菊花花蕾激光顯微切割樣品RNA提取質量的影響。［方法］以丙酮和乙醇冰醋酸2種試劑固定菊花花蕾，采用石蠟和冰凍切片2種方式制作樣本，顯微切割并收集最外兩輪小花原基，提取RNA后比較提取質量。［結果］石蠟和經梯度蔗糖保護的冰凍切片均能很好地保存花蕾組織形態，就石蠟切片而言，乙醇冰醋酸固定組織切割樣品的RNA降解明顯低于丙酮固定樣品；而2種固定劑的冰凍切片切割樣品RNA提取質量無明顯差異。與石蠟切片相比，冰凍切片顯微切割樣品RNA質量更高。切割時收集管蓋加入RNA提取緩沖液可明顯緩解RNA降解。顯微切割的不同品種菊花花原基與花發育有關的基因表達水平存在差異，表明切割獲得的材料可用于下一步研究。［結論］植物組織經乙醇冰醋酸固定、梯度蔗糖保護后冰凍切片進行顯微切割效果較佳，可富集特異組織或細胞并從中獲得較高質量的RNA。

關鍵詞 固定劑；石蠟切片；冰凍切片；激光顯微切割；RNA；提取質量

中圖分類號 Q94-336 文獻標識碼 A

文章編號 0517-6611（2024）18-0001-05

doi：10.kXl6dXLwb1b/JPHinal3W4GmMrEUNvbXyQ434Q+AeFg=3969/j.issn.0517-6611.2024.18.001

開放科學（資源服務）標識碼（OSID）：

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

MA Yue-hua，DING Lian，ZHANG Xue et al

（College of Horticulture，Nanjing Agricultural University，Nanjing，Jiangsu 210095）

Abstract ［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.

Key words Fixative；Paraffin section；Cryosection；Laser microdissection；RNA；Extraction quality

基金項目 中央高校基本業務費科技平臺實驗技術人才基金項目（10307201705）。

作者簡介 馬月花（1975—），女，青海海東人，高級實驗師，博士，從事實驗室和大型儀器管理工作。

收稿日期 2023-10-19

激光顯微切割技術是通過在顯微鏡下識別目的組織或細胞，利用激光作為能量，從不同的組織中快速切割、分離和純化生物體的目標組織、細胞，從而進行后續的分子生物學、質譜等分析的一種手段，它是顯微觀察和分子生物學、代謝組學等研究之間的橋梁。……