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 highthroughput studies.

A Technical Note on Quantum Dots for Multi-Color Fluorescence in situ Hybridization

“…Their use in FISH experiments has indeed been investigated for several years [Pathak et al, 2001;Xiao and Barker, 2004]; however, surprisingly few reports on the successful application of Qdots can be found in the literature, and only recently Qdot-conjugated antibodies against haptens (for example digoxigenin or dinitrophenyl) or against biotin have become commercially available. Until today, successful visualization of FISH probes using Qdots has been reported for repetitive sequences [Pathak et al, 2001;Wu et al, 2006;Ma et al, 2008], for gene-specific probes [Xiao and Barker, 2004;Jiang et al, 2007] and for oligonucleotide probes [Byers et al, 2007;Knoll, 2007], but so far not for the delineation of entire chromosome territories by chromosome painting.…”

Identification of CD44v6+/CD24− breast carcinoma cells in primary human tumors by quantum dot-conjugated antibodies

“…12,14 The imaging system comprised a Leitz Diaplan fluorescence microscope (Leitz, Germany) with a 490-nm excitation filter and a CRI Nuance spectral analyzer (Cambridge Research and Instrumentation Inc., Woburn, MA, USA). The CRI Nuance analyzer contains a liquid crystal tunable filter (LCTF) and a CCD (charged-coupled device) camera.…”

“…Several antigens or mRNAs can then be assessed in a single tissue section using multiple quantum dots with different emission spectra. [12][13][14] The signals from the quantum dots are deconvoluted by spectral unmixing to generate a composite image of each signal. Spectral unmixing also separates autofluorescence from quantum dot signals, an important step when studying formalin-fixed, paraffin-embedded (FFPE) samples.…”

Quality Assurance in Her-2 Testing—Redefining the Gold Standard

“…Such variation can lead to one reference laboratory adjudging a case as unequivocally negative and another laboratory adjudging the case as unequivocally positive. The development of new fluorescence-based techniques for both protein and gene-copy detection will allow the introduction of truly quantitative and robust testing, with a real potential for automation and testing on a large scale (13 ). Underpinning this testing regimen with PCR-verified tissue will produce confidence in the testing laboratories by clinical colleagues and patients alike.…”