Single-molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

Jan Hesse; Jaroslaw Jacak; Gerhard Regl; Thomas Eichberger; Fritz Aberger; Robert Schlapak; Stefan Howorka; Leila Muresan; Anna-Maria Frischauf; Gerhard J. Schütz

doi:10.1117/12.700244

14 February 2007 Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

Jan Hesse, Jaroslaw Jacak, Gerhard Regl, Thomas Eichberger, Fritz Aberger, Robert Schlapak, Stefan Howorka, Leila Muresan, Anna-Maria Frischauf, Gerhard J. Schütz

Author Affiliations +

Proceedings Volume 6444, Ultrasensitive and Single-Molecule Detection Technologies II; 64440F (2007) https://doi.org/10.1117/12.700244
Event: SPIE BiOS, 2007, San Jose, California, United States

Abstract

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm² size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10⁶ cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

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Jan Hesse, Jaroslaw Jacak, Gerhard Regl, Thomas Eichberger, Fritz Aberger, Robert Schlapak, Stefan Howorka, Leila Muresan, Anna-Maria Frischauf, and Gerhard J. Schütz "Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling", Proc. SPIE 6444, Ultrasensitive and Single-Molecule Detection Technologies II, 64440F (14 February 2007); https://doi.org/10.1117/12.700244

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