title: Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.
abstract: OBJECTIVE
  Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues.
  
  
  METHODS
  Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR.
  
  
  RESULTS
  No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility.
  
  
  CONCLUSIONS
  For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560-568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3.
authors.affiliation: Department of Orthopaedic Surgery, VU University Medical Center (VUMC), Center for Translational Regenerative Medicine (CTRM), MOVE Research Institute Amsterdam, Amsterdam, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Peeters
authors.affiliation: Kolling Institute, Sydney Medical School - Northern, Royal North Shore Hospital, University of Sydney,Sydney, Australia.
authors.forename: C L
authors.initials: CL
authors.lastname: Huang
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: L A
authors.initials: LA
authors.lastname: Vonk
authors.affiliation: Tissue Engineering and Biomaterials Research Unit, Sydney University, Sydney, Australia.
authors.forename: Z F
authors.initials: ZF
authors.lastname: Lu
authors.affiliation: Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: R A
authors.initials: RA
authors.lastname: Bank
authors.affiliation: Department of Orthopaedic Surgery, VU University Medical Center (VUMC), Center for Translational Regenerative Medicine (CTRM), MOVE Research Institute Amsterdam, Amsterdam, The Netherlands m.helder@vumc.nl.
authors.forename: M N
authors.initials: MN
authors.lastname: Helder
authors.affiliation: Department of Orthopaedic Surgery, VU University Medical Center (VUMC), Center for Translational Regenerative Medicine (CTRM), MOVE Research Institute Amsterdam, Amsterdam, The Netherlands.
authors.forename: B Zandieh
authors.initials: BZ
authors.lastname: Doulabi
keywords: Annulus fibrosus; Articular cartilage; Meniscus; Nucleus pulposus; Real-time polymerase chain reaction; Ribonucleic acid; Ribonucleic acid isolation