Efficient DNA/RNA extraction from tarsal plates by SK mill, a freeze-crush apparatus. (26th September 2022)

• Record Type: Journal Article
• Authors: Oku, Hiroaki; Watanabe, Akihide; Tokuda, Yuichi; Nakayama, Tomomichi; Tashiro, Kei; Sotozono, Chie; Nakano, Masakazu
• Is Part Of: Journal of biochemistry. Volume 172:Number 6(2022)
• Page Start: 385
• Page End: 394
• Publication Date: 2022-09-26
• Subjects: DNA/RNA extraction -- eyelid sebaceous carcinoma -- freeze crush -- next-generation sequencer -- tarsal plate; Biochemistry -- Periodicals; Biochemistry -- Periodicals; Electronic journals; 572.05
• DOI: 10.1093/jb/mvac078
• Languages: English
• ISSNs: 0021-924X

Abstract

Abstract: 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. Graphical Abstract: