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


RESULTS
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.
Ruettger
Anke
A
Institute of Molecular Pathogenesis (IMP), Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Jena, Germany. anke.ruettger@fli.bund.de.
Neumann
Steffi
S
Wiederanders
Bernd
B
Huber
René
R