RNA Isolation from Articular Cartilage Tissue

Hongjun Zheng, Audrey McAlinden

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Isolation of high-quality RNA directly from tissues is desirable to obtain precise information of in vivo gene expression profiles in cells embedded within their extracellular matrix (ECM). It is well known that purification of RNA from cartilage tissues is particularly challenging due to low cell (chondrocyte) content and its dense ECM rich in large negatively charged proteoglycans that can copurify with RNA. Older methodologies to purify RNA from cartilage involved the use of concentrated denaturing solutions containing guanidinium isothiocyanate followed by ultracentrifugation in cesium trifluoroacetate. Such ultracentrifugation approaches are rarely used now since the emergence of more user-friendly mini spin column chromatography kits. For this chapter, we tested and compared three methods to isolate RNA from immature murine articular (femoral head) cartilage and found that the combination of TRIzol® reagent and spin column chromatography (Norgen Total RNA Purification Kit) was the best approach to generate higher quality RNA. Here, the average RNA Integrity Number (RIN), as determined by Bioanalyzer technology, was 7.1. We then applied this method to attempt to isolate RNA directly from human articular cartilage harvested from three osteoarthritic (OA) knee joint specimens. As expected, the concentration and quality of RNA obtained differed between samples. However, from one specimen, we were able to isolate approximately 3 μg of total RNA (including small noncoding RNAs) from 100 mg of human OA cartilage with a RIN = 7.9. Despite the patient-to-patient variabilities that are known to exist between cartilage specimens from OA joints, we have demonstrated that it is possible to obtain reasonably high levels of RNA from human OA articular cartilage at a quality suitable for downstream analyses including microarray and RNA-Seq. A detailed description of our preferred RNA purification methodology, which can be used to isolate RNA from human, bovine, or rodent cartilage tissue, is provided in this chapter.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages121-133
Number of pages13
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2245
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Articular cartilage
  • Bioanalyzer
  • Cartilage
  • Chondrocyte
  • NanoDrop™
  • Norgen kit
  • Osteoarthritis
  • RNA
  • RNA purification
  • Spin column
  • TRIzol
  • Tissue pulverization

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