A method for obtaining high integrity RNA from developing aleurone cells and starchy endosperm in rice (Oryza sativa L.) by laser microdissection
A method for obtaining high integrity RNA from developing aleurone cells and starchy endosperm in rice (Oryza sativa L.) by laser microdissection
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DOI：
10.1016/j.plantsci.2007.06.004
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发表时间：
2007-09-01
期刊：
影响因子：
5.2
通讯作者：
Kondo, Motohiko
中科院分区：
文献类型：
--
作者：
Ishimaru, Tsutomu;Nakazono, Mikio;Kondo, Motohiko
激光显微切割(LMD)正成为阐明基因表达空间图谱的有力工具。然而，一个仍然存在的困难是从固定和嵌入的组织中获得高完整性的RNA，以便获得准确和可靠的表达分析。本研究旨在建立利用LMD技术从发育中的水稻胚乳的糊粉细胞和淀粉胚乳中提取高完整性RNA的方法。固定剂为丙酮或3：1乙醇：醋酸(AA)，包埋剂为2%羧甲基纤维素(CMC)或石蜡。对于细胞类型的鉴定和完整RNA的回收，AA和CMC更好，尽管CMC包埋不像石蜡包埋那样保存形态。定量RT-PCR结果表明，编码蔗糖转运蛋白的OsSUTI基因存在于糊粉细胞中，编码淀粉去分支酶(普鲁兰酶)的SDBE基因存在于淀粉胚乳中。综上所述，这些结果表明我们的LMD方法适合于从水稻糊粉细胞和淀粉胚乳中提取高完整性的RNA。用这种方法获得的mRNA有助于了解胚乳发育的分子基础。(C)2007爱思唯尔爱尔兰有限公司。保留所有权利。
Laser microdissection (LMD) is now becoming a powerful tool to elucidate the spatial profiling of gene expression. However, a remaining difficulty is obtaining high integrity RNA from fixed and embedded tissues in order to obtain accurate and reliable expression analyses. This study aimed to develop methods for the preparation of high integrity RNA from aleurone cells and starchy endosperm of developing rice endosperm by LMD. Acetone or 3:1 ethanol:acetic acid (AA), was used for the fixative and 2% carboxymethyl cellulose (CMC) or paraffin, was used for the embeddina reaLent. AA and CMC were better for identification of cell types and for recovery of intact RNA, although CMC-embedding did not preserve morphology as well as paraffin-embedding. Quantitative RT-PCR revealed that OsSUTI mRNA encoding a sucrose transporter was localized in aleurone cells and that SDBE mRNA encoding a starch debranching enzyme (pullulanase) was localized in starchy endosperm. Taken together, these results show that our LMD method is suitable for preparing high integrity RNA from aleurone cells and starchy endosperm in rice. mRNA obtained with this method should help to understand the molecular basis for endosperm development. (c) 2007 Elsevier Ireland Ltd. All rights reserved.