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

Abstract

Background: The 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.

Findings: We 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(R)/RNeasy Mini kit), working in a reproducible and reliable manner.

Conclusions: We 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.

Figure 1

Comparison of current methods for total RNA isolation from the small cell tissue cartilage: quality control using capillary electrophoresis. Total RNA was isolated from chondrocytes (cells) and cartilage explants. Cartilage homogenization was performed with scalpel (SC), rotor-stator (RS) or microdismembrator (MD). Different extraction procedures (TRIzol^®, RNeasy™, TRIzol^®/RNeasy™ and RNAqueous™) were performed as described in the Methods section. Integrity of RNA isolated from different species (human, adult bovine cartilage - cow, and immature bovine cartilage - calf) was analyzed by capillary electrophoresis. 18S and 28S rRNA bands correspond to 41-43 and 47-50 [s], respectively. The RNA yield is specified for each sample [in ng/μl]. After precipitation and washing the RNA was resuspended in different volumes of RNase-free water: 30 μl (Trizol^®), 20 μl (RNeasy™), 10 - 20 μl (Trizol^®/RNeasy™) and 10 - 20 μl (RNAqueous™). On account of this, the results provided here are comparable with the results provided in Table 1 (μg RNA per 100 mg cartilage). The results are shown by gel electrophoresis (RIN: RNA integrity number; N/A: not available).