Efficient DNA/RNA extraction from tarsal plates by SK mill, a freeze-crush apparatus
Efficient DNA/RNA extraction from tarsal plates by SK mill, a freeze-crush apparatus
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使用冷冻粉碎设备 SK mill 从跗骨板中高效提取 DNA/RNA
DOI：
10.1093/jb/mvac078
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Nakano Masakazu
中科院分区：
文献类型：
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作者：
Oku Hiroaki;Watanabe Akihide;Tokuda Yuichi;Nakayama Tomomichi;Tashiro Kei;Sotozono Chie;Nakano Masakazu
睑板是从上/下眼睑内部维持眼睑结构的眼睑组织，并且它围绕睑板腺并支持其独特的分泌机制。皮脂腺癌是一种恶性眼睑肿瘤，有时会从睑板腺发展而来，通常在手术过程中与睑板一起切除，因此睑板作为对照研究组织。然而，由于板厚、硬且异质且细胞很少，因此通常难以获得足够的基因组DNA和/或总RNA。因此，我们试图建立一个有效的协议，以获得DNA和RNA的同时，通过比较匀浆（研钵/杵，丸杵或SK磨）和纯化（有机溶剂或旋转柱）的方法，使用兔跗板的组合。根据产量、质量和操作时间，SK轧机和纺丝塔被认为是最有效的组合。然后，我们应用所建立的方案从六个人睑板样本中提取DNA/RNA，并通过下一代测序仪成功地生成高质量的外显子组和转录组数据集，分别具有足够的覆盖率和代表性基因的睑板腺特异性表达。我们的新发现将为今后皮脂腺癌的遗传学和基因表达研究提供理想的参考数据。
The tarsal plate is an eyelid tissue that maintains lid structure from inside the upper/lower eyelids, and it surrounds the meibomian glands and supports their unique secretion mechanism. Sebaceous carcinoma, a malignant eyelid tumour, can sometimes develop from the meibomian glands and is usually excised together with the tarsal plate during surgery, so the tarsal plate serves as a control research tissue. However, since the plate is thick, hard and heterogeneous with few cells, obtaining enough genomic DNA and/or total RNA is often difficult. Therefore, we attempted to establish an efficient protocol to obtain DNA and RNA simultaneously by comparing the combinations of homogenization (mortar/pestle, pellet pestle or SK mill) and purification (organic solvent or spin column) methods using rabbit tarsal plates. Based on the yield, quality and hands-on time, the SK mill and spin column was found to be the most efficient combination. We then applied the established protocol to extract DNA/RNA from six human tarsal-plate samples and succeeded in generating high-quality exome and transcriptome datasets via a next-generation sequencer with sufficient coverage and meibomian gland-specific expression of representative genes, respectively. Our new findings will provide ideal reference data for future genetic and gene-expression studies of sebaceous carcinoma.