Креативная хирургия и онкология (Jul 2020)
Non-Coding RNA Extraction from Nucleus Pulposus of Intervertebral Discs with Subsequent Expression Profiling
Abstract
Background. With numerous extraction protocols for total RNA and RNA fractions, like microRNA (miRNA) and long non-coding RNA (lncRNA), available for various cell and tissue types, obtaining a high quantitative and qualitative yield from some special material, such as the nucleus pulposus, remains challenging. Nucleus pulposus is troublesome to manage in common RNA isolation protocols due to low cell content and high biopolymer concentrations, including proteoglycans and glycoproteins, which impair overall purity and yield. A major lack of reproducible methods for total and fraction RNA isolation directly from the nucleus pulposus impedes effective real-time PCR applications for downstream miRNA and lncRNA expression profiling in the course of intervertebral disc degeneration. In this study, we exploit the collagenase type II lytic properties to facilitate extraction of total and fraction RNA from the nucleus pulposus and compare results with the standard RNA isolation method.Materials and methods. Nucleus pulposus samples (n = 8) were obtained from September 2017 to December 2018 from patients with herniated discs in the lumbosacral spine diagnosed during surgery. Equal portions of samples were processed with the standard and original RNA isolation protocols.Results and discussion. Th e enzymatic lysis method for total and fraction RNA isolation from the nucleus pulposus of intervertebral discs demonstrated excellent integrity and high purity. No protein, polysaccharide or collagen contamination was detected.Conclusion. Th e method reported allows an improved quantitative and qualitative total and fraction RNA yield from the nucleus pulposus of intervertebral discs. Th e method can be used in future research on miRNA and lncRNA expression profiling with real-time PCR by improving the average cycle threshold value.
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