Comparison of different methods for preparation and characterization of total RNA from cartilage samples to uncover osteoarthritis in vivo

Abstract

BackgroundThe isolation of intact RNA can be very difficult when tissues are used that contain many RNAses or that are hard to homogenize, e.g. cartilage samples. Additionally, cartilaginous tissues are characterized by a low cellularity and an abundance of extracellular matrix (ECM) molecules. But given the growing interest in understanding pathogenesis of degenerative diseases, e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), studies have to consider expression pattern of cells in its natural environment.FindingsWe compared the current RNA isolation methods for the extraction of high-quality RNA of snap-frozen biopsies from limited amounts of hypocellular cartilaginous tissue. The focus of the study was to gather information about procedure-related differences in RNA quality and yield. Here, we describe two protocols, the phenol/chloroform-free filter-based method (RNAqueous™ kit) and the combined protocol (TRIzol®/RNeasy Mini™ kit), working in a reproducible and reliable manner.ConclusionsWe conclude that preparation, storage, homogenization, and quality control are altogether critical steps for in-depth analysis of differential gene expression, especially in hypocellular tissues with highly crosslinked ECM like cartilage.

The initial repair response of articular cartilage after mechanically induced damage

“…Liquid nitrogen frozen cartilage specimens from the center (Ø3 mm, tissue wet weight 8.5 ± 2.5 mg) of the loaded samples ( n = 6) and static controls ( n = 3) were reduced to a fine powder in a mikro‐dismembrator (Sartorius–Stedim Biotech, Nieuwegein, The Netherlands). Total RNA was extracted from the homogenized samples according to a protocol described by Ruettger et al using the RNA queous Midi™ kit (Life Technologies, Bleiswijk, The Netherlands). Concentration and purity of the RNA was measured by a NanoDrop spectrophotometer (Isogen, De Meern, The Netherlands) at 260 and 280 nm.…”

Comparison of various commercial products for phenol-guanidine-based classical swine fever virus RNA extraction

“…Whereas numerous studies have been published which compare different RNA extraction procedures, both for cellular (Ruettger et al, 2010;Kong et al, 2006) and for viral (Deng et al, 2005;Guarino et al, 1997;Scheibner et al, 2000) RNA extraction, no published data are available on the comparison of various commercial products that are all based on the phenol-guanidine principle. In the present study we compare the performance of TRIzol ® with several other brand name products based on the same principle for the extraction of CSFV RNA from cell culture supernatant, serum and tonsils.…”

Extraction of high-quality RNA from human articular cartilage

“…Although conventional RNA extraction protocols and kits are routinely used to purify high-quality RNA from different cell lines and tissues, preparing high-quality RNA from particular tissues remains challenging. Articular cartilage is among the tissues that challenge conventional RNA extraction protocols due to its low cellularity and high proteoglycan content that compromise RNA purity and yield [2, 3]. Furthermore, the rigid and dense matrix structure of the articular cartilage necessitates harsh homogenization conditions, which compromise RNA integrity [2, 3].…”

“…Articular cartilage is among the tissues that challenge conventional RNA extraction protocols due to its low cellularity and high proteoglycan content that compromise RNA purity and yield [2, 3]. Furthermore, the rigid and dense matrix structure of the articular cartilage necessitates harsh homogenization conditions, which compromise RNA integrity [2, 3]. Accordingly, obtaining cartilage RNA with an A 260/280 ratio ∼1.8, an A 260/230 ratio ∼2, and a RIN value ∼7 has never been achieved with any of the thus far available protocols [2, 3].…”

“…Furthermore, the rigid and dense matrix structure of the articular cartilage necessitates harsh homogenization conditions, which compromise RNA integrity [2, 3]. Accordingly, obtaining cartilage RNA with an A 260/280 ratio ∼1.8, an A 260/230 ratio ∼2, and a RIN value ∼7 has never been achieved with any of the thus far available protocols [2, 3]. Enzymatic digestion methods that isolate chondrocytes from articular cartilage tissues prior to RNA extraction have been developed to overcome this problem [4].…”