Perocchi Fabiana, Xu Zhenyu, Clauder-Münster Sandra, Steinmetz Lars M
European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Nucleic Acids Res. 2007;35(19):e128. doi: 10.1093/nar/gkm683. Epub 2007 Sep 26.
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.
最近的转录谱研究表明，在真核生物和细菌基因组中，反义转录的比例出人意料地高。然而，反义转录本的范围和意义存在争议，部分原因是怀疑存在实验假象。在这里，我们提出了一种在逆转录过程中使用放线菌素D（ActD）来生成全基因组范围内纯净转录组图谱的方法。我们表明，反义假象似乎是由逆转录反应中第二链cDNA的假合成引发的。比较了在有和没有ActD的情况下制备的样品中从酿酒酵母平铺阵列获得的链特异性杂交信号。ActD的使用去除了约一半可检测到的反义转录本，这与其为假象一致，而正义表达水平和约200个反义转录本不受影响。因此，我们的发现有助于更准确地评估反义转录的范围和位置，以便更好地理解其在细胞中的作用。
