Method for the Isolation of "RNA-seq-Quality" RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization.
Cells
; 11(22)2022 11 11.
Article
in En
| MEDLINE
| ID: mdl-36429007
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Cartilage, Articular
/
Intervertebral Disc
Limits:
Humans
Language:
En
Journal:
Cells
Year:
2022
Document type:
Article
Affiliation country:
RUSSIA