genotype: Wild-type phenotype: Tick Field strain not resistant to acaricide sample type: Brahman adult tick developmental stage: adult
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction RNA was prepared from whole frustrated larvae, and adult ticks collected as described above. The ticks were first ground in liquid nitrogen using a sterile mortar and pestle. Total RNA was isolated using the TRIzol® reagent according to the manufacturer’s protocol (GibcoBRL, USA). Total RNA was also prepared by the same procedure from 20 000 unattached/unfed larvae (L1 and L2). The mRNA was purified from these samples using the Poly (A) PuristTM MAG Kit (AMBION, USA) as recommended by the manufacturer. cDNA Synthesis cDNA was prepared from the above mRNA samples using the SuperScriptTM Double-Stranded cDNA Synthesis Kit (Invitrogen, USA) as recommended by the manufacturer with the exception that after the second strand cDNA synthesis is terminated by the addition of 0.5 M EDTA and before the phenol: chloroform: isoamyl alcohol step, a RNase A treatment step was included as recommended in the NimbleGen cDNA protocol (NimbleGen, USA). After this RNase A treatment the Superscript double stranded cDNA Synthesis protocol was followed as described in the technical manual. cDNA median size was verified by 1% agarose electrophoresis in TAE 1X, and 2 microg of each cDNA sample were sent to NimbleGen Systems Inc. (Madison, WI, USA) for microarray hybridization using the R. microplus custom array (NimbleGen Custom Design name: 2006-05-22_B_microplus_50mer_exp).
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
17 days old (soon after the 2nd tick moult from nymph to adult stages) approximately 500 young adult female ticks were collected from the Brahman cattle A total of six tick naïve cattle, three Brahman and three Holstein-Friesian female cattle were infested with 1.5 g (~30,000) N strain larvae [31] and were kept grazing at the University of Queensland’s Pinjarra Hills campus. On Day 2, approximately 20,000 larvae were placed into a 24 cm2 mesh bag and attached to the neck of each animal for approx. 5 hrs in order for the larvae to ‘sense’ host stimuli while also in the presence of other attached ticks. These ‘frustrated’ larvae from both the Brahman (B-FL) and Holstein-Friesian (H-FL) were subsequently frozen in liquid nitrogen for total RNA extraction. An additional 20,000 unattached larvae (L - unfed) were processed to provide a control larvae group without host stimulus. At 17 days (soon after the 2nd tick moult from nymph to adult stages) approximately 500 young adult female ticks were collected from the Brahman and Holstein-Friesian cattle and frozen for total RNA extraction. The sampling regime was repeated to provide biological replicates of each sample to improve the statistical significance of the gene expression data (1 and 2 denote biological replicates). The experimental samples were: unattached/unfed larvae: L1, L2. “Frustrated” larvae: B-FL1; B-FL2 and HFL1; HFL 2. Attached adult female ticks (~17 days): – B-AT1, B-AT2 and H-AT1, H-AT2. (B=Brahman; H=Holstein-Friesian; FL=frustrated larvae; AT=adult tick)
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
