Emerging Genome Technologies
Eveland, Andrea L , Kirst, Matias , Latshaw, Susan P , Avigne, W.T. , McCarty, Donald R , Koch, Karen E .
3’UTR Profiling: Resolution of Differential Expression by Closely-Related Transcripts and Identification of Novel Sequences.
The specificity and information content of 3’UTRs is here harnessed in a novel approach to transcriptome profiling. Results are also compared to microarray analyses. We describe a 3’-anchoring strategy for sequence-based profiles using 454 technology. By specifically targeting the 3’UTR of mRNAs, quantitative resolution was achieved for differential expression of Near-Identical Transcripts (NITs). These can include specific alleles, paralogs, or gene-family members. In addition, multiplex adaptors, each modified with a 4-base recognition key, can be used to concurrently sequence and identify up to 48 individual samples. We compared 3’UTR profiles of maize ovaries from wild-type and an ABA-insensitive mutant in response to drought. Resolution of closely-related transcripts identified instances of differential expression. Contrasting responses were indicated between mutant and wild-type plants, for example, by read frequencies (quantitative measure of mRNAs) for two Auxin Repressed Dormancy Associated NITs. Specific amplification of the 3’UTR by Q-PCR confirmed this differential response for the closely-related ARDA transcripts to ABA-based sensing. We further assessed the potential for 454-based 3’UTR profiling in global transcriptome analysis in parallel with a microarray approach. A multiplexed library of 12 samples representing 4 sequential stages of maize ovary development generated ~250,000 good-quality reads in a single 454 “run”. Resolution of differential expression between NITs and quantitative profiles of transcriptional changes provided an invaluable complement to microarray analyses. In addition, recovery of newly-encountered, non-arrayed genes enabled flexibility for gene discovery in a non-sequenced genome.
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1 - University of Florida, Department of Horticultural Sciences; UF Genetics Institute, 1301 Fifield Hall, Gainesville, FL, 32611, USA
2 - University of Florida, Department of Forest Resources and Conservation; UF Genetics Institute
3 - University of Florida, Department of Horticultural Sciences; UF Genetics Institute
4 - University of Florida, Department of Horticultural Sciences
Presentation Type: ASPB Minisymposium
Location: Continental A/Hilton
Date: Sunday, July 8th, 2007
Time: 2:00 PM