Artemii A. IvanovNovosibirsk state university, Russia
Title: Method of isolation RNA for transcriptome analysis from human intervertebral discs
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 cartilage recommend the use of expensive equipment, enzymatic matrix digestion, or cell culture.
We have developed a method to isolate high-quality RNA from postsurgical material of human intervertebral discs, which only requires mortar and pestle, TRIzol and RNEasy spin columns. First, a sample is to be grinded in the liquid nitrogen. An awl is used to break the sample into pieces, then they are hammered repeatedly with a mortar. The resulting particles are grinded into fine powder and mixed with the frozen TRIzol to prevent degradation during the thawing.
Next, the sample undergoes two consecutive phase separations followed by the final column extraction step. This combination allows to dispose of proteoglycans and obtain RNA pure enough for the RNA-seq. 32 samples have been processed this far with success rate of 81.25%. Mean RNA integrity number and concentration of samples which were sent for sequencing were 7.7 ± 0.15 and 46.9 ± 8.9 ng/ul respectively.
RNA-seq analysis of the sequenced 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.
Our protocol is designed to minimize both the equipment requirements and the use of invasive methods that might alter the expression profile. It could be used to collect transcriptome data from intervertebral discs worldwide.
The work was supported by the Russian Scientific Foundation (RSF) grant ?22-15-20037.
Artemii Ivanov completed his Bachelor’s degree at Novosibirsk state university in 2022. He is now a first-year master’s student at NSU. He has already published three articles as the first author and developed a novel protocol for the efficient isolation of RNA from human intervertebral discs.