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

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.

Arabidopsis MSI1 connects LHP1 to PRC2 complexes

“…Leaf number 6 was harvested at ZT ( zeitgeber time)=7 h, and RNA was isolated, labelled using the GeneChip WT Sense Target Labeling Assay and hybridized to Affymetrix AGRONOMICS1 Arabidopsis tiling arrays as described (Rehrauer et al , 2010; Müller et al , 2012). Data were normalized and analysed as described (Rehrauer et al , 2010; Müller et al , 2012), based on TAIR10 annotations (http://www.arabidopsis.org). Leaf‐specific expression potentials were estimated as the maximum expression measured in any of the leaf samples from the AtGenExpress reference set for development (Schmid et al , 2005).…”

“…Plants were grown for 48 days in short‐day photoperiods. Leaf number 6 was harvested at ZT ( zeitgeber time)=7 h, and RNA was isolated, labelled using the GeneChip WT Sense Target Labeling Assay and hybridized to Affymetrix AGRONOMICS1 Arabidopsis tiling arrays as described (Rehrauer et al , 2010; Müller et al , 2012). Data were normalized and analysed as described (Rehrauer et al , 2010; Müller et al , 2012), based on TAIR10 annotations (http://www.arabidopsis.org).…”

Transgenerational phenotype aggravation in CAF‐1 mutants reveals parent‐of‐origin specific epigenetic inheritance

“…The experiment was performed in biological triplicates with each triplicate consisting of pooled leaves from five, seven and nine plants for WT Col, fas2-4 G1 and fas2-4 G6, respectively. Data were normalized and analyzed as described (Rehrauer et al, 2010;M€ uller et al, 2012). Differential expression was tested with LIMMA (Smyth, 2004) followed by multiple testing correction (Storey & Tibshirani, 2003).…”

Distinct modes of DNA accessibility in plant chromatin

“…Summarized gene transcript abundances were derived using the Robust Multi-array Average (RMA) algorithm as described 53 . Summarized gene antisense transcript abundances were derived similarly, using the probes from the antisense strand 60 . Strand-specific RNA-seq data were obtained from Gene Expression Omnibus (GEO) (accession code GSM277612 (ref.…”