Multi-targeted priming for genome-wide gene expression assays

• Main Authors: Adomas Aleksandra B, Lopez-Giraldez Francesc, Clark Travis A, Wang Zheng, Townsend Jeffrey P
• Format: Article
• Language: English
• Published: BMC 2010-08-01
• Series: BMC Genomics
• Online Access: http://www.biomedcentral.com/1471-2164/11/477

<p>Abstract</p> <p>Background</p> <p>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 <it>Saccharomyces cerevisiae </it>and of <it>Neurospora crassa</it>, 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.</p> <p>Results</p> <p>We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of <it>Saccharomyces cerevisiae </it>and <it>Neurospora crassa </it>while avoiding priming ribosomal RNA or transfer RNA. Examining the response of <it>Saccharomyces cerevisiae </it>to nitrogen deficiency and profiling <it>Neurospora crassa </it>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.</p> <p>Conclusions</p> <p>Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and <it>N. crassa </it>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.</p>