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Proceedings Paper

Single-molecule fluorescence microscopy for ultra-sensitive RNA expression profiling
Author(s): Jan Hesse; Jaroslaw Jacak; Gerhard Regl; Thomas Eichberger; Fritz Aberger; Robert Schlapak; Stefan Howorka; Leila Muresan; Anna-Maria Frischauf; Gerhard J. Schütz
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Paper 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.

Paper Details

Date Published: 14 February 2007
PDF: 7 pages
Proc. SPIE 6444, Ultrasensitive and Single-Molecule Detection Technologies II, 64440F (14 February 2007); doi: 10.1117/12.700244
Show Author Affiliations
Jan Hesse, Upper Austrian Research GmbH (Austria)
Jaroslaw Jacak, Johannes Kepler Univ. Linz (Austria)
Gerhard Regl, Univ. Salzburg (Austria)
Thomas Eichberger, Univ. Salzburg (Austria)
Fritz Aberger, Univ. Salzburg (Austria)
Robert Schlapak, Upper Austrian Research GmbH (Austria)
Stefan Howorka, Upper Austrian Research GmbH (Austria)
Leila Muresan, Johannes Kepler Univ. Linz (Austria)
Anna-Maria Frischauf, Univ. Salzburg (Austria)
Gerhard J. Schütz, Johannes Kepler Univ. Linz (Austria)

Published in SPIE Proceedings Vol. 6444:
Ultrasensitive and Single-Molecule Detection Technologies II
Jorg Enderlein; Zygmunt K. Gryczynski, Editor(s)

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