Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D - PubMed (original) (raw)

Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D

Fabiana Perocchi et al. Nucleic Acids Res. 2007.

Abstract

Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.

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Figures

Figure 1.

Figure 1.

Hypothetical models for spurious second-strand synthesis by reverse transcription (upper panel) and the mode of inhibition by ActD (lower panel). During sample processing first-strand cDNA is generated from RNA molecules by reverse transcription. Spurious second-strand cDNA synthesis could occur using the first-strand cDNA as a template. Two possible mechanisms are: self-priming through hairpin loop extension of the first-strand cDNA, or re-priming either from RNA fragments formed during degradation of the RNA templates or from primers present in the reaction. In both cases, hybridization to strand-specific arrays would lead to artifactual antisense signals. ActD inhibits second-strand synthesis (lower panel, red cross), putatively by binding to bases of single-stranded DNA molecules, and therefore artifactual antisense-strand signals would not be detected.

Figure 2.

Figure 2.

Genome-wide analysis of sense and antisense signals from hybridizations performed in either the absence (ActD−) or presence (ActD+) of ActD during reverse transcription. (A) Dependence of antisense signal intensity on the sense expression level. Red curves represent the fitted hybridization signals using smoothing splines. (B) Variation in signal intensities among biological replicates for the sense and antisense strands (SD). (C) Two examples of spurious antisense transcripts resolved by ActD. (D) Two examples of consistent antisense transcripts unaffected by ActD. In both panels C, and D, normalized signal intensities are shown for probes along their chromosomal position using a color gradient (_x_-axis; W, Watson strand; C, Crick strand). Upper panel, five ActD− replicates (_y_-axis, ActD−, 1–5); lower panel, three ActD+ replicates (_y_-axis, ActD+ 1–3). Profiles for all genomic regions are available online. (

www.ebi.ac.uk/huber-srv/actinomycinD

).

Figure 3.

Figure 3.

Scatter plot of segment expression levels between arrays generated with and without ActD. Green dots, antisense segments detected above background in ActD− samples; Red line, identical expression in both conditions; dashed lines, background thresholds.

Figure 4.

Figure 4.

Validation of antisense transcripts. (A) Comparison of ActD and computational filtering. Expression level of antisense segments in reactions with (ActD+) and without ActD (ActD−). For ActD− data, only antisense segments which passed the computational filter are shown. The dotted lines represent the background thresholds. Blue dots, segments common between ActD+ and filtered ActD−; orange dots, unique to ActD+; green dots, unique to filtered ActD−. A list of these segments is available (Supplementary Table 4). (B) Strand-specific RT-PCR of antisense transcripts. Three antisense RNAs opposite to MRB1, EPL1 and MRK1 yielded a RT-PCR product. Seven regions for which putative antisense artifacts were detected by array analysis opposite of CYS4, EMP24, PNC1, MDH3, HAC1, TRR1 and PFK1 did not yield a RT-PCR product. ACT1 sense RT-PCR reactions served as internal controls for each individual RT-PCR; –RT designate RT reactions in which the reverse transcriptase was omitted; gDNA indicates PCR reactions with genomic DNA as a template; reactions were performed in triplicate.

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