High quality RNA isolation from tumours with low cellularity and high extracellular matrix component for cDNA microarrays: application to chondrosarcoma
- Department of Pathology, Leiden University Medical Centre, PO Box 9600, L1-Q, 2300 RC Leiden, The Netherlands
- Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, Shikata, Japan
- Professor Hogendoorn P.C.W.Hogendoorn{at}lumc.nl
- Accepted 10 July 2001
Abstract
Aims—High quality RNA isolation from cartilaginous tissue is considered difficult because of relatively low cellularity and the abundance of extracellular matrix rich in glycosaminoglycans and collagens. Given the growing interest and technical possibilities to study RNA expression at a high throughput level, research on tissue with these characteristics is hampered by the lack of an efficient method for obtaining sufficient amounts of high quality RNA.
Methods—This paper presents a robust protocol combining two RNA isolation procedures, based on a combination of Trizol and RNA specific columns, which has been developed to obtain high molecular weight RNA from fresh frozen and stored tissue of normal cartilage and cartilaginous tumours. Using this method, RNA was isolated from normal cartilage, peripheral chondrosarcoma, and central chondrosarcoma.
Results—The yields ranged from 0.1 to 0.5 μg RNA/mg tissue. RNA isolated with this method was stable and of high molecular weight. RNA samples from normal cartilage and from two chondrosarcomas isolated using this method were applied successfully in cDNA microarray experiments. The number of genes that give interpretable results was in the range of what would be expected from microarray results obtained on chondrosarcoma cell line RNA. Signal to noise ratios were good and differential expression between tumour and normal cartilage was detectable for a large number of genes.
Conclusion—With this newly developed isolation method, high quality RNA can be obtained from low cellular tissue with a high extracellular matrix component. These procedures can also be applied to other tumour material.
