RNAlater facilitates microdissection of sensory cell-enriched samples from the mouse cochlea for transcriptional analyses.
RNAlater facilitates microdissection of sensory cell-enriched samples from the mouse cochlea for transcriptional analyses.
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DOI：
10.1016/j.jneumeth.2013.08.010
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发表时间：
2013-10-15
影响因子：
3
通讯作者：
Hu, Bo Hua
中科院分区：
文献类型：
--
作者：
Cai, Qunfeng;Wang, Bo;Patel, Minal;Yang, Shi Ming;Hu, Bo Hua
耳蜗病理学的分子分析依赖于高质量耳蜗样本的采集。对于小型啮齿类动物来说，分离出富含感觉细胞且 RNA 完整性保存完好的样品进行转录分析是一项重大挑战。在这里，我们报告了一种从耳蜗中分离富含感觉细胞的样本的显微解剖技术。我们发现用 RNAlater（一种 RNA 保存介质）处理组织可以改变组织的物理特性并有利于解剖。与之前从感觉上皮分离的样品不同，我们的样品含有确定的细胞群，其感觉细胞与支持细胞的比例一致。重要的是，RNA 成分保存完好。通过这种显微解剖方法，我们收集了三种类型的样本：富含感觉细胞、富含外毛细胞和富含内毛细胞的样本。为了证明该方法的可行性，我们在富含感觉细胞的样本中筛选了多个参考基因，并在噪声损伤的耳蜗中鉴定了稳定的基因。这里描述的方法平衡了对感觉细胞质量和纯度的需求，并且还规避了目前收集耳蜗组织技术的许多限制。通过我们的方法，收集的样本可用于各种下游分析，包括 qRT-PCR、微阵列和 RNA 测序。
Molecular analyses of cochlear pathology rely on the acquisition of high-quality cochlear samples. For small rodents, isolating sensory cell-enriched samples with well-preserved RNA integrity for transcriptional analyses poses a significant challenge. Here, we report a microdissection technique for isolating sensory cell-enriched samples from the cochlea. We found that treating the tissue with RNAlater, a RNA preservation medium, alters the physical properties of the tissue and facilitates the dissection. Unlike previous samples that have been isolated from the sensory epithelium, our samples contain defined cell populations that have a consistent ratio of sensory cells to supporting cells. Importantly, the RNA components were well preserved. With this microdissection method, we collected three types of samples: sensory cell-enriched, outer hair cell-enriched, and inner hair cell-enriched. To demonstrate the feasibility of the method, we screened multiple reference genes in the sensory cell-enriched samples and identified stable genes in noise-traumatized cochleae. The method described here balances the need for both quality and purity of sensory cells and also circumvents many limitations of the currently-available techniques for collecting cochlear tissues. With our approach, the collected samples can be used in diverse downstream analyses, including qRT-PCR, microarray, and RNA sequencing.
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影响因子：
6.1
作者：
Cai, Qunfeng;Patel, Minal;Coling, Donald;Hu, Bo Hua
通讯作者：
Hu, Bo Hua
影响因子：
2.8
作者：
Hu, BH;Henderson, D;Nicotera, TM
通讯作者：
Nicotera, TM
DOI：
10.1007/bf00339444
发表时间：
1961-01-01
期刊：
ZEITSCHRIFT FUR ZELLFORSCHUNG UND MIKROSKOPISCHE ANATOMIE
影响因子：
--
作者：
IURATO, S
通讯作者：
IURATO, S
影响因子：
4
作者：
Towers, Emily R.;Kelly, John J.;Dawson, Sally J.
通讯作者：
Dawson, Sally J.
影响因子：
3
作者：
Anderson, Charles T.;Zheng, Jing
通讯作者：
Zheng, Jing