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

Artemii A. Ivanov, Olga N. Leonova, Daniil S. Wiebe, Alexsandr V. Krutko, Mariya M. Gridina, Veniamin S. Fishman, Yurii S. Aulchenko, Yakov A. Tsepilov, Tatiana S. Golubeva

Результат исследования: Научные публикации в периодических изданияхстатьярецензирование

Аннотация

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.

Язык оригиналаанглийский
Номер статьи3578
ЖурналCells
Том11
Номер выпуска22
DOI
СостояниеОпубликовано - нояб. 2022

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