Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®.
Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®.
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DOI：
10.1371/journal.pone.0066854
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Sternberg LR
中科院分区：
文献类型：
--
作者：
Golubeva YG;Smith RM;Sternberg LR
激光显微切割是医学研究中的宝贵工具，有助于收集特定细胞群进行分子分析。然而，研究目标的多样性（例如临床和动物研究中的癌性和癌前病变、细胞沉淀、啮齿动物胚胎等）和不同的科学目标对建立标准激光显微切割方案提出了挑战。样品制备对于高质量 RNA、DNA 和蛋白质检索至关重要，它通常决定激光显微切割项目的可行性。临床和动物模型研究中的大多数显微解剖研究都是在含有未经固定修饰的天然核酸的冷冻组织上进行的。然而，含有脂肪、胶原蛋白或脆弱细胞结构的组织冷冻切片的形态质量变化可能会限制或阻止通过激光解剖成功收获所需细胞群。据报道，CryoJane Tape-Transfer System® 是一种可改善载玻片冷冻切片结果的商业设备，在激光解剖过程中非常适合载玻片制备和高质量骨细胞 RNA（冷冻骨）的分离。考虑到报道的 CryoJane 在载玻片激光切割方面的优势，我们询问该系统是否也可以与基于紫外激光的显微切割仪器使用的塑料膜载玻片配合使用，因为这些载玻片更适合收集更大的目标区域。为了优化不同 RNA 稳定性和冷冻切片难度的组织的激光显微切割载玻片制备，我们评估了 CryoJane 系统与玻璃（激光捕获显微切割）和膜（激光切割显微切割）载玻片的使用。我们建立了玻璃和膜载玻片的样品制备方案，包括用 CryoJane 溶液手动涂覆膜载玻片、冷冻切片、载玻片染色和解剖程序、裂解和 RNA 提取，以促进从 CryoJane 制剂中进行高效解剖和高质量 RNA 检索。因此，CryoJane 技术有潜力促进冷冻组织激光显微切割载玻片制备的标准化。
Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues.
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影响因子：
6.9
作者：
Matas AJ;Agustí J;Tadeo FR;Talón M;Rose JK
通讯作者：
Rose JK
通过对叶绿体和光合细菌之间代谢网络的比较分析来探索光合作用的演变。
影响因子：
4.4
作者：
Wang, Zhuo;Zhu, Xin-Guang;Chen, Yazhu;Li, Yuanyuan;Hou, Jing;Li, Yixue;Liu, Lei
通讯作者：
Liu, Lei
影响因子：
6
作者：
Schwaninger, Ruth;Rentsch, Cyrill A.;Cecchini, Marco G.
通讯作者：
Cecchini, Marco G.
影响因子：
4.1
作者：
Ehrig, T;Abdulkadir, SA;Watson, MA
通讯作者：
Watson, MA
影响因子：
--
作者：
Kube DM;Savci-Heijink CD;Lamblin AF;Kosari F;Vasmatzis G;Cheville JC;Connelly DP;Klee GG
通讯作者：
Klee GG