Multi-targeted priming for genome-wide gene expression assays.
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.
Adomas
Aleksandra B
AB
Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511, USA.
Lopez-Giraldez
Francesc
F
Clark
Travis A
TA
Wang
Zheng
Z
Townsend
Jeffrey P
JP
D017931:DNA Primers / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics*; D053858:Metabolic Networks and Pathways / Q000187:drug effects / Q000235:genetics; D025282:Mycelium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D009492:Neurospora crassa / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D009584:Nitrogen / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048348:Reverse Transcription / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017423:Sequence Analysis, RNA
D017931:DNA Primers; D012331:RNA, Fungal; D012333:RNA, Messenger; D009584:Nitrogen