Automated brightfield break-apart in situ hybridization (ba-ISH) application

ALK and MALT1 genes as models

Hiroaki Nitta, Wenjun Zhang, Brian D. Kelly, Melanie Miller, Lidija Pestic-Dragovich, Christopher Bieniarz, Thomas J. Vasicek, Teresa Marafioti, Lisa M Rimsza, Thomas M. Grogan

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5′ and 3′ probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.

Original languageEnglish (US)
Pages (from-to)352-358
Number of pages7
JournalMethods
Volume52
Issue number4
DOIs
StatePublished - Dec 2010

Fingerprint

Marginal Zone B-Cell Lymphoma
Protein Transport
In Situ Hybridization
Genes
Tissue
Fluorescence In Situ Hybridization
Proteins
Fluorescence
Lymphoid Tissue
Mucous Membrane
Digoxigenin
Genetic Translocation
Microscopes
Neoplasm Genes
Palatine Tonsil
Photobleaching
Paraffin
Formaldehyde
Alkaline Phosphatase
anaplastic lymphoma kinase

Keywords

  • ALK
  • Automation
  • Break-apart probes
  • Brightfield
  • Gene translocation
  • In situ hybridization
  • Lymphoma
  • MALT1

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Nitta, H., Zhang, W., Kelly, B. D., Miller, M., Pestic-Dragovich, L., Bieniarz, C., ... Grogan, T. M. (2010). Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models. Methods, 52(4), 352-358. https://doi.org/10.1016/j.ymeth.2010.07.005

Automated brightfield break-apart in situ hybridization (ba-ISH) application : ALK and MALT1 genes as models. / Nitta, Hiroaki; Zhang, Wenjun; Kelly, Brian D.; Miller, Melanie; Pestic-Dragovich, Lidija; Bieniarz, Christopher; Vasicek, Thomas J.; Marafioti, Teresa; Rimsza, Lisa M; Grogan, Thomas M.

In: Methods, Vol. 52, No. 4, 12.2010, p. 352-358.

Research output: Contribution to journalArticle

Nitta, H, Zhang, W, Kelly, BD, Miller, M, Pestic-Dragovich, L, Bieniarz, C, Vasicek, TJ, Marafioti, T, Rimsza, LM & Grogan, TM 2010, 'Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models', Methods, vol. 52, no. 4, pp. 352-358. https://doi.org/10.1016/j.ymeth.2010.07.005
Nitta, Hiroaki ; Zhang, Wenjun ; Kelly, Brian D. ; Miller, Melanie ; Pestic-Dragovich, Lidija ; Bieniarz, Christopher ; Vasicek, Thomas J. ; Marafioti, Teresa ; Rimsza, Lisa M ; Grogan, Thomas M. / Automated brightfield break-apart in situ hybridization (ba-ISH) application : ALK and MALT1 genes as models. In: Methods. 2010 ; Vol. 52, No. 4. pp. 352-358.
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abstract = "Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5′ and 3′ probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.",
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