Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Lambros, Maryou B. K., Simpson, Peter T., Jones, Chris, Natrajan, Rachael, Westbury, Charlotte, Steele, Dawn, Savage, Kay, Mackay, Alan, Schmitt, Fernando C., Ashworth, Alan and Reis-Filho, Jorge S. (2006) Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization. Laboratory Investigation, 86 4: 398-408. doi:10.1038/labinvest.3700390


Author Lambros, Maryou B. K.
Simpson, Peter T.
Jones, Chris
Natrajan, Rachael
Westbury, Charlotte
Steele, Dawn
Savage, Kay
Mackay, Alan
Schmitt, Fernando C.
Ashworth, Alan
Reis-Filho, Jorge S.
Title Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization
Journal name Laboratory Investigation   Check publisher's open access policy
ISSN 0023-6837
Publication date 2006-01
Sub-type Article (original research)
DOI 10.1038/labinvest.3700390
Volume 86
Issue 4
Start page 398
End page 408
Total pages 11
Editor J. Crawford
Place of publication London
Publisher Nature Publishing
Collection year 2006
Language eng
Subject C1
321020 Pathology
730108 Cancer and related disorders
Abstract Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.
Keyword Medicine, Research & Experimental
Pathology
Chromogenic In Situ Hybridization
Fluorescent In Situ Hybridization
Comparative Genomic Hybridization
Molecular Pathology
Protocol
Comparative Genomic Hybridization
Secretory Breast-carcinoma
Lobular Carcinoma
Array-cgh
Amplification
Cancer
Tumors
Dna
Her2
Expression
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
2007 Higher Education Research Data Collection
School of Medicine Publications
 
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Created: Wed, 15 Aug 2007, 07:56:45 EST