Email updates

Keep up to date with the latest news and content from BMC Molecular Biology and BioMed Central.

Open Access Highly Accessed Methodology article

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

Wen-Han Yu12, Hedda Høvik3, Ingar Olsen3 and Tsute Chen1*

Author Affiliations

1 Department of Molecular Genetics, The Forsyth Institute, Cambridge, MA, USA

2 Bioinformatics Graduate Program, Boston University, Boston, MA, USA

3 Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway

For all author emails, please log on.

BMC Molecular Biology 2011, 12:3  doi:10.1186/1471-2199-12-3

Published: 14 January 2011

Abstract

Background

With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles.

Results

RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts.

Conclusions

An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment.