title: Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries.
abstract: BACKGROUND
  Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions.
  
  
  RESULTS
  Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions.
  
  
  CONCLUSIONS
  Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.
authors.affiliation: Department of Biological Sciences, Plymouth State University, MSC 64, 17 High St,, Plymouth, NH 03264, USA. katieroseboissonneault@gmail.com.
authors.forename: Katie Rose
authors.initials: KR
authors.lastname: Boissonneault
authors.forename: Brooks M
authors.initials: BM
authors.lastname: Henningsen
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Bates
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Robertson
authors.forename: Sean
authors.initials: S
authors.lastname: Milton
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Hogan
authors.forename: David E
authors.initials: DE
authors.lastname: Housman
mesh_terms: D017377:Diatoms / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D004398:Dyneins / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006657:Histones / Q000235:genetics; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008387:Marine Toxins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D006657:Histones; D008387:Marine Toxins; D004398:Dyneins; C012301:domoic acid; D007608:Kainic Acid