Connective Tissue Oncology Society

2001 CTOS Annual Meeting Posters— Biology

HIGH QUALITY RNA ISOLATION FROM CHONDROSARCOMA; APPLICATION TO CDNA MICROARRAYS
H. J. Baelde¹, A. M. Cleton-Jansen¹, H. van Beerendonk¹, M. Namba², J. V. Bovee¹, P. C. Hogendoorn¹
¹Department of Pathology, Leiden University Medical Center, ²Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School

OBJECTIVE: High quality RNA isolation from cartilaginous tissue is considered difficult due to relative low cellularity and the abundance of extracellular matrix rich in glycosaminoglycans and collagen. Given the growing interest and technical possibilities to study RNA expression at a high throughput level research on certain tumour types is hampered because of aforementioned characteristics.

METHODS: We present a robust method using a combination of two RNA isolation procedures that has been developed to obtain high molecular weight RNA from fresh frozen and stored tissue of normal cartilage and cartilaginous tumors. Using this method RNA was isolated from normal cartilage, peripheral and central chondrosarcoma and from chondrosarcoma cell lines SW1353 and OUMS-27. RNA quality was validated after electrophoresis and staining with ethidium bromide. Subsequent conversion to cDNA and labelling was followed by application to a Micromax Human cDNA microarray system and fluorescence intensity was scanned using an Affimetrix/GMS 418 scanner and analysed with a GenePix Pro 3.0 analysis program.

RESULTS: The yields range from 0.1-0.5 g RNA per mg tissue. 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 is in the range of what is expected when compared with microarray results obtained on chondrosarcoma cell line RNA. Signal-to-noise ratios are good and differential expression between tumor and normal cartilage is detectable for a large number of genes.

CONCLUSION: With this newly developed isolation method high quality RNA can be obtained from low cellular tissue whith high extracellular matrix component. This RNA is of suitable quality for subsequent cDNA microarray studies. These procedures can be applied to other difficult tumor material as well.