Transcription profiling of Strongyloides ratti parasitic females experiencing an anti-S. ratti low or high imumne response measured at 15 or 6 days of infection

ABSTRACT: We wished to determine the changes in gene expression that occurred in S. ratti parasitic females as an infection progressed and thus as these stages are exposed to an anti-S. ratti immune response. To do this we compared gene expression in parasitic females recovered 6 days p.i. (i.e. no or very low immune response) with those recovered at 15 days p.i. (i.e. high immune response); for convenience, we refer to these as parasitic females subject to "low immune pressure" and "high immune pressure", respectively. These days were chosen because previous analyses of S. ratti parasitic females have shown significant differences in the size, appearance etc. of worms at these time points. The experimental design used, was to have at least three biological replicates for each sample (i.e. three independent preparations of the relevant worm samples and their RNA) and to have at least three technical replicates (i.e. independent, separate cDNA synthesis, amplification and hybridization etc.) for each biological replicate. For each hybridisation (below) a dye-swap was used i.e. each sample to be used in a hybridisation was labelled, separately, with each of the two dyes (below).

21,085 ESTs were sequenced from various S. ratti stage-specific libraries, of which 14,761 resulted in sequence data above a quality threshold that were then submitted to public databases. 11,551 clones were derived from the S. ratti parasitic libraries of which 7,385 produced sequence data (above a quality threshold); all of these 7,385 clones were arrayed together with a random sample of 1,619 of clones for which no sequence data were available. These 7,385 ESTs are highly redundant since they represent 2,963 contigs and 2,125 clusters, both including 1,220 singletons (i.e. clusters or contigs containing only one EST). Notwithstanding this redundancy, they were used in the microarray construction for two reasons: (i) this approach was less error-prone than attempting to select a unique clone set and (ii) this in-built redundancy provides many replicates of individual contigs and clusters, which can be exploited in quality-control analyses. In addition to these 9,004 S. ratti clones, the following controls were included: 281 EST clones from the mixed iL3/free-living adult library (representing 173 contigs and 167 clusters, 67 of which are singletons) to ensure that gene expression in the parasitic and free-living stages could be differentiated; 230 commercially available controls (Amersham Biosciences UK, Ltd.). 12 poly-A and 457 spotting buffer-only controls. Thus, in total 9,984 spots were arrayed.

INSTRUMENT(S): Axon- GenePix4000B

ORGANISM(S): Strongyloides ratti

DISEASE(S): hign immune pressure

SUBMITTER: Fiona Thompson 

PROVIDER: E-MEXP-1010 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress