title: Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies.
abstract: Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.
authors.affiliation: University Department of Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK.
authors.forename: Eleni
authors.initials: E
authors.lastname: Tholouli
authors.forename: Judith A
authors.initials: JA
authors.lastname: Hoyland
authors.forename: Dolores
authors.initials: D
authors.lastname: Di Vizio
authors.forename: Fionnuala
authors.initials: F
authors.lastname: O'Connell
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Macdermott
authors.forename: David
authors.initials: D
authors.lastname: Twomey
authors.forename: Richard
authors.initials: R
authors.lastname: Levenson
authors.forename: John A Liu
authors.initials: JA
authors.lastname: Yin
authors.forename: Todd R
authors.initials: TR
authors.lastname: Golub
authors.forename: Massimo
authors.initials: M
authors.lastname: Loda
authors.forename: Richard
authors.initials: R
authors.lastname: Byers
mesh_terms: D001706:Biopsy; D005404:Fixatives; D005557:Formaldehyde / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D045663:Quantum Dots; D012333:RNA, Messenger / Q000032:analysis*
chemical_list: D005404:Fixatives; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D005557:Formaldehyde