title: Semiautomated multiplexed quantum dot-based in situ hybridization and spectral deconvolution.
abstract: Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to high-throughput studies.
authors.affiliation: Division of Laboratory and Regenerative Medicine, University of Manchester, United Kingdom.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Byers
authors.forename: Dolores
authors.initials: D
authors.lastname: Di Vizio
authors.forename: Fionnuala
authors.initials: F
authors.lastname: O'connell
authors.forename: Eleni
authors.initials: E
authors.lastname: Tholouli
authors.forename: Richard M
authors.initials: RM
authors.lastname: Levenson
authors.forename: Kirk
authors.initials: K
authors.lastname: Gossage
authors.forename: Kirk
authors.initials: K
authors.lastname: Gossard
authors.forename: David
authors.initials: D
authors.lastname: Twomey
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.forename: Elisa
authors.initials: E
authors.lastname: Benedettini
authors.forename: Joshua
authors.initials: J
authors.lastname: Rose
authors.forename: Keith L
authors.initials: KL
authors.lastname: Ligon
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Finn
authors.forename: Todd R
authors.initials: TR
authors.lastname: Golub
authors.forename: Massimo
authors.initials: M
authors.lastname: Loda
mesh_terms: D000818:Animals; D019070:Cell Lineage; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes; D045663:Quantum Dots; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D015345:Oligonucleotide Probes