1. Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization
- Author
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Artemii A. Ivanov, Olga N. Leonova, Daniil S. Wiebe, Alexsandr V. Krutko, Mariya M. Gridina, Veniamin S. Fishman, Yurii S. Aulchenko, Yakov A. Tsepilov, and Tatiana S. Golubeva
- Subjects
Cartilage, Articular ,Chondrocytes ,cartilage RNA isolation ,RNA-seq ,RIN ,intervertebral disc ,Humans ,RNA ,General Medicine ,RNA-Seq ,Intervertebral Disc - Abstract
The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture. In our study, we used a combination of the traditional QIAzol protocol (Qiagen, Germany) and RNEasy column purification (Qiagen, Germany) to obtain high-quality RNA from post-surgical intervertebral disc fragments. Only a mortar and a pestle were used for grinding, making our method particularly accessible. The isolated RNA with a RIN of ~7 is suitable for studying the expression profile of chondrocytes in situ. RNA-seq analysis of three samples demonstrated cell type ratios to be mostly relevant to intervertebral disc tissues, with over 70% of the chondrocytes of the three subtypes having an admixture of blood-related cells.
- Published
- 2022
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