• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jclinpathJournal of Clinical PathologyCurrent TOCInstructions for authors
J Clin Pathol. Oct 2001; 54(10): 778–782.
PMCID: PMC1731295

High quality RNA isolation from tumours with low cellularity and high extracellular matrix component for cDNA microarrays: application to chondrosarcoma

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.

Key Words: RNA extraction • cartilage • bone neoplasm • chondrosarcoma • cDNA microarray • expression profile

Full Text

The Full Text of this article is available as a PDF (189K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet. 1996 Dec;14(4):457–460. [PubMed]
  • Snijders AM, Meijer GA, Brakenhoff RH, van den Brule AJ, van Diest PJ. Microarray techniques in pathology: tool or toy? Mol Pathol. 2000 Dec;53(6):289–294. [PMC free article] [PubMed]
  • Wildsmith SE, Elcock FJ. Microarrays under the microscope. Mol Pathol. 2001 Feb;54(1):8–16. [PMC free article] [PubMed]
  • Bovée JV, Cleton-Jansen AM, Kuipers-Dijkshoorn NJ, van den Broek LJ, Taminiau AH, Cornelisse CJ, Hogendoorn PC. Loss of heterozygosity and DNA ploidy point to a diverging genetic mechanism in the origin of peripheral and central chondrosarcoma. Genes Chromosomes Cancer. 1999 Nov;26(3):237–246. [PubMed]
  • Bovée JV, van Royen M, Bardoel AF, Rosenberg C, Cornelisse CJ, Cleton-Jansen AM, Hogendoorn PC. Near-haploidy and subsequent polyploidization characterize the progression of peripheral chondrosarcoma. Am J Pathol. 2000 Nov;157(5):1587–1595. [PMC free article] [PubMed]
  • Bovée JV, Cleton-Jansen AM, Wuyts W, Caethoven G, Taminiau AH, Bakker E, Van Hul W, Cornelisse CJ, Hogendoorn PC. EXT-mutation analysis and loss of heterozygosity in sporadic and hereditary osteochondromas and secondary chondrosarcomas. Am J Hum Genet. 1999 Sep;65(3):689–698. [PMC free article] [PubMed]
  • Bovée JV, van den Broek LJ, Cleton-Jansen AM, Hogendoorn PC. Up-regulation of PTHrP and Bcl-2 expression characterizes the progression of osteochondroma towards peripheral chondrosarcoma and is a late event in central chondrosarcoma. Lab Invest. 2000 Dec;80(12):1925–1934. [PubMed]
  • Kunisada T, Miyazaki M, Mihara K, Gao C, Kawai A, Inoue H, Namba M. A new human chondrosarcoma cell line (OUMS-27) that maintains chondrocytic differentiation. Int J Cancer. 1998 Sep 11;77(6):854–859. [PubMed]
  • van Gijlswijk RP, Zijlmans HJ, Wiegant J, Bobrow MN, Erickson TJ, Adler KE, Tanke HJ, Raap AK. Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem. 1997 Mar;45(3):375–382. [PubMed]
  • Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, et al. Molecular portraits of human breast tumours. Nature. 2000 Aug 17;406(6797):747–752. [PubMed]
  • McCormick C, Duncan G, Tufaro F. New perspectives on the molecular basis of hereditary bone tumours. Mol Med Today. 1999 Nov;5(11):481–486. [PubMed]
  • Bernard MA, Hogue DA, Cole WG, Sanford T, Snuggs MB, Montufar-Solis D, Duke PJ, Carson DD, Scott A, Van Winkle WB, et al. Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations. J Bone Miner Res. 2000 Mar;15(3):442–450. [PubMed]
  • Legeai-Mallet L, Rossi A, Benoist-Lasselin C, Piazza R, Mallet JF, Delezoide AL, Munnich A, Bonaventure J, Zylberberg L. EXT 1 gene mutation induces chondrocyte cytoskeletal abnormalities and defective collagen expression in the exostoses. J Bone Miner Res. 2000 Aug;15(8):1489–1500. [PubMed]

Figures and Tables

An external file that holds a picture, illustration, etc.
Object name is 01150.f1.jpg
Figure 1 Example of RNA isolated from chondrosarcoma run on a formaldehyde/agarose gel and stained with ethidium bromide. 28S and 18S ribosomal RNA bands are indicated.
An external file that holds a picture, illustration, etc.
Object name is 01150.f2.jpg
Figure 2 Example of cDNA microarray hybridised with primary tumour RNA of a central chondrosarcoma and normal cartilage.

Articles from Journal of Clinical Pathology are provided here courtesy of BMJ Group

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...