Cargando…

Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Ivanov, Artemii A., Leonova, Olga N., Wiebe, Daniil S., Krutko, Alexsandr V., Gridina, Mariya M., Fishman, Veniamin S., Aulchenko, Yurii S., Tsepilov, Yakov A., Golubeva, Tatiana S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9688557/
https://www.ncbi.nlm.nih.gov/pubmed/36429007
http://dx.doi.org/10.3390/cells11223578
Descripción
Sumario: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.