TY - JOUR

T1 - Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization

AU - Ivanov, Artemii A.

AU - Leonova, Olga N.

AU - Wiebe, Daniil S.

AU - Krutko, Alexsandr V.

AU - Gridina, Mariya M.

AU - Fishman, Veniamin S.

AU - Aulchenko, Yurii S.

AU - Tsepilov, Yakov A.

AU - Golubeva, Tatiana S.

N1 - Funding Information: A.S.D.: Financial support was received from P30 CA008748 (National Cancer Institute, National Institutes of Health). Funding Information: This work was supported by the Russian Science Foundation (RSF) grant No. 22-15-20037 and by the Government of the Novosibirsk region. Funding Information: Authors A.S.D. and M.J.B. declare no competing interests. Author C.G.M. has received speaker (Illumina and Amgen) and consultant (Faze, Beam) honoraria, and receives research funding from AbbVie and Pfizer. Publisher Copyright: © 2022 by the authors.

PY - 2022/11

Y1 - 2022/11

N2 - 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.

AB - 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.

KW - cartilage RNA isolation

KW - intervertebral disc

KW - RIN

KW - RNA-seq

KW - RNA/metabolism

KW - Intervertebral Disc

KW - RNA-Seq

KW - Humans

KW - Chondrocytes/metabolism

KW - Cartilage, Articular/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85142416305&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/2f56d1a7-cc23-3722-94f4-4c54005a3a0e/

U2 - 10.3390/cells11223578

DO - 10.3390/cells11223578

M3 - Article

C2 - 36429007

AN - SCOPUS:85142416305

VL - 11

JO - Cells

JF - Cells

SN - 2073-4409

IS - 22

M1 - 3578

ER -