Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 M E T H O D O L O G Y A R T I C L E Open Access Multi-targeted priming for genome-wide gene expression assays Aleksandra B Adomas1, Francesc Lopez-Giraldez1, Travis A Clark1, Zheng Wang1, Jeffrey P Townsend2* Abstract Background: Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. Results: We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Conclusions: Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome. Background Gene expression levels have been quantified by numer- ous procedures, including reverse transcription (RT)- PCR [1], sequencing of expressed sequence tags [2], serial analysis of gene expression [3], microarray hybri- dization [4], and massively parallel signature sequencing [5]. Rapid development of platforms has improved throughput, but also generated strong demand for enhanced sensitivity and measurement accuracy. For nearly all expression assays, reverse transcription from * Correspondence: Jeffrey.Townsend@Yale.edu 2Department of Ecology and Evolutionary Biology, Program in Computational Biology and Bioinformatics, and Microbiology Graduate Program, Yale University, 165 Prospect St, New Haven, CT 06511, USA Full list of author information is available at the end of the article messenger RNA (mRNA) to complementary DNA (cDNA) is a key step of the process that contributes less experimental variance than biological growth and har- vest, but greater experimental variance than hybridiza- tion [[6], but see also [7]]. Throughput of the reaction may be biased by secondary and tertiary structures of mRNA, affinities specific to the reverse transcriptase, inhibitors present in the sample, priming strategy, and variation in priming efficiency [8]. The most common priming strategies utilize oligo-dT primers, random pri- mers, or gene-specific primers. When oligo-dT primers are used for reverse transcription, RNA secondary struc- ture and variation in poly(A) tail length may result in gene amplification 3′ bias [9]. Random primers, typically used in prokaryotic systems, fail to discriminate between © 2010 Adomas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.