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Open Access Methodology

Evaluation of alternative RNA labeling protocols for transcript profiling with Arabidopsis AGRONOMICS1 tiling arrays

Marlen Müller1, Andrea Patrignani2, Hubert Rehrauer2, Wilhelm Gruissem12 and Lars Hennig13*

Author Affiliations

1 Department of Biology, Plant Biotechnology, ETH Zurich, Zurich, Switzerland

2 Functional Genomics Center Zurich, ETH and University of Zurich, CH-8057, Zurich, Switzerland

3 Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO-Box 7080, SE-75007, Uppsala, Sweden

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Plant Methods 2012, 8:18  doi:10.1186/1746-4811-8-18

Published: 13 June 2012

Abstract

Microarrays are routine tools for transcript profiling, and genomic tiling arrays such as the Arabidopsis AGRONOMICS1 arrays have been found to be highly suitable for such experiments because changes in genome annotation can be easily integrated at the data analysis level. In a transcript profiling experiment, RNA labeling is a critical step, most often initiated by oligo-dT-primed reverse transcription. Although this has been found to be a robust and reliable method, very long transcripts or non-polyadenylated transcripts might be labeled inefficiently. In this study, we first provide data handling methods to analyze AGRONOMICS1 tiling microarrays based on the TAIR10 genome annotation. Second, we describe methods to easily quantify antisense transcripts on such tiling arrays. Third, we test a random-primed RNA labeling method, and find that on AGRONOMICS1 arrays this method has similar general performance as the conventional oligo-dT-primed method. In contrast to the latter, however, the former works considerably better for long transcripts and for non-polyadenylated transcripts such as found in mitochondria and plastids. We propose that researchers interested in organelle function use the random-primed method to unleash the full potential of genomic tiling arrays.

Keywords:
Transcript profiling; Arabidopsis; Microarray; AGRONOMICS1; Tiling array; RNA labeling