Efficient DNA/RNA extraction from tarsal plates by SK mill, a freeze-crush apparatus
Autor: | Hiroaki Oku, Akihide Watanabe, Yuichi Tokuda, Tomomichi Nakayama, Kei Tashiro, Chie Sotozono, Masakazu Nakano |
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Rok vydání: | 2022 |
Předmět: | |
Zdroj: | The Journal of Biochemistry. 172:385-394 |
ISSN: | 1756-2651 0021-924X |
Popis: | 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. |
Databáze: | OpenAIRE |
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