Transcriptome sequencing and microarray development for the woodrat (Neotoma spp.): custom genetic tools for exploring herbivore ecology.

Massively parallel sequencing has enabled the creation of novel, in-depth genetic tools for nonmodel, ecologically important organisms. We present the de novo transcriptome sequencing, analysis and microarray development for a vertebrate herbivore, the woodrat (Neotoma spp.). This genus is of ecological and evolutionary interest, especially with respect to ingestion and hepatic metabolism of potentially toxic plant secondary compounds. We generated a liver transcriptome of the desert woodrat (Neotoma lepida) using the Roche 454 platform. The assembled contigs were well annotated using rodent references (99.7% annotation), and biotransformation function was reflected in the gene ontology. The transcriptome was used to develop a custom microarray (eArray, Agilent). We tested the microarray with three experiments: one across species with similar habitat (thus, dietary) niches, one across species with different habitat niches and one across populations within a species. The resulting one-colour arrays had high technical and biological quality. Probes designed from the woodrat transcriptome performed significantly better than functionally similar probes from the Norway rat (Rattus norvegicus). There were a multitude of expression differences across the woodrat treatments, many of which related to biotransformation processes and activities. The pattern and function of the differences indicate shared ecological pressures, and not merely phylogenetic distance, play an important role in shaping gene expression profiles of woodrat species and populations. The quality and functionality of the woodrat transcriptome and custom microarray suggest these tools will be valuable for expanding the scope of herbivore biology, as well as the exploration of conceptual topics in ecology.

Changes in gene expression induced by chemoradiation in advanced cervical carcinoma: a microarray study of RTOG C-0128.

PURPOSE: To evaluate gene expression patterns in patients with advanced cervix cancer before and during chemoradiation in a multi-institutional cooperative group setting. METHODS: RTOG C0128 was designed as a Phase II trial of radiation therapy with concomitant chemotherapy and Celecoxib at 400 mg twice daily for one year. Tumor samples were obtained for microarray gene expression analysis before treatment and at the time of the first implant (paired sample). RNA was extracted, linearly amplified, and purity was assessed by gel electrophoresis. Each sample was hybridized against a universal RNA mixture on a customized spotted array consisting of >10,000 genes. Gene expression pre-treatment was compared with clinical characteristics. Changes in gene expression following radiation were assessed within the paired samples (same patient) and then compared across all paired samples. Data were normalized using the AROMA software, and clustering analysis was performed using Ward's method in Spotfire. Differences in paired samples were calculated with Significance Analysis of Microarrays (SAM). RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial. Tissue was obtained prior to initiation of therapy from 34 patients (40%). FIGO stages of the patients providing tissue were IB (23%), II (57%), and IIIA-IVA (20%). RNA quality was sufficient in 22 pre-treatment and 14 post-treatment samples. Among pre-treatment samples, no significant differences in gene expression were observed by FIGO stage, age, or race. However, between comparison of histologic subtypes (adenocarcinoma, n=5; squamous cell carcinoma, n=17) demonstrated 45 genes differentially expressed with a false discovery rate of 0.018. Cluster analysis segregated unpaired samples into 2 groups: 18/22 comprising pre-treatment samples and 10/14 in group 2 representing post-treatment samples. In all 13 paired samples, gene expression after chemoradiation was significantly upregulated in 91 genes and downregulated in 251 genes (false discovery rate of 0.0018). Genes significantly upregulated included bax, cdk inhibitor 1, MMP2, and adhesion molecules PECAM1, VCAM1, and ICAM2. Genes significantly downregulated included topoisomerase II alpha, myc, H2AX, MSH2, RAD51, RAD53, PCNA, and cell cycle-regulating molecules chk1, CDK2, cyclinB1, cyclin D3, cdc2, and cdc25. CONCLUSIONS: Microarray analysis was successfully performed in a multi-institutional cooperative group trial. Gene expression significantly correlated with histology, but not stage, age or race. Cluster analysis identified two groups of gene expression profiles correlating with pre or post-treatment acquisition of tissue. Notably, paired samples showed significant changes in gene expression following chemoradiation, including several downregulated radiation response genes. Further analysis comparing gene expression to clinical outcomes, acute and late toxicities awaits maturation of clinical data. Hopefully, this data will lead to the development of molecularly based therapies.