genotype: Wild-type phenotype: Tick Field strain not resistant to acaricide treatment group: dsRNA injected developmental stage: adult Sex: female
Growth protocol
NFRS (non-resistant field strain) adult female ticks were collected from infested cattle from the Department of Employment, Economic Development & Innovation’s (formerly Dept. of Primary Industries & Fisheries) Biosecurity tick colony kindly provided by Dr Louise Jackson (Stewart et al., 1982). Fresh adult female ticks were collected at 21 days fed to repletion and were utilised on the day of collection in microinjection experiments. The 600bp dsRNA targeting TC6372 (R. microplus Ubiquitin-63E homologue) was prepared as described previously (Kurscheid et al 2009) using PCR amplification and T7 RNA transcription as per manufacturer’s instructions (Ambion MEGAScript RNAi kit, Applied Biosystems, CA, USA). Thirty ticks were utilised to obtain RNA for each microarray slide. Adult female ticks were injected via the spiracle with 5µL of 3330nM of dsRNA and PBS injected controls (30 ticks per experimental duplicate, total 120 ticks). All ticks were incubated in a humidified incubator at 28oC (Kurscheid et al., 2009). Ticks were inspected daily for mortalities and oviposition and after 5 days were removed for RNA processing. After rinsing in 0.1% DEPC-treated water, ticks were dissected in an 8cm culture dish (Nalgene) kept in a tray of wet ice. Sterile scalpel blades and forceps were pre-treated with RNaseZAP (Ambion, CA, USA). An incision was made with a sterile razor blade just above the right spiracle, starting at the right side of the capitulum and cutting around to the left side of the capitulum. The dorsal cuticle was then lifted with a pair of dissection forceps. Viscera were individually transferred into 2mL screw-cap microfuge tubes containing 1.5mL ice-cold TRIzol reagent (Invitrogen, CA, USA) and 100mg of 1mm sterile glass beads. The viscera were then homogenized using a bead beater for 5 minutes prior to RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the TRIzol® reagent according to the manufacturer’s protocol (GibcoBRL, USA). The RNA pellet was re-suspended in 400µL 0.1% DEPC-treated water. The total RNA was examined for integrity using gel electrophoresis, and subsequently stored at -80°C until further use. cDNA was prepared from the RNA extracted from un-treated controls and the injected Ubiquitin-63E (TC6372) 600bp dsRNA treated ticks above (prepared in duplicate from 30 ticks treated and un-treated). mRNA was prepared 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 was terminated by the addition of 0.5 M EDTA, and, prior to the phenol:chloroform:isoamyl alcohol extraction, a RNase A treatment step was included as recommended in the NimbleGen cDNA protocol (NimbleGen, USA) (Rodriguez Valle et al., 2010). 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 g 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
dsRNA injected adult female ticks
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.).
Microarray evidence for off target effects and the optimisation of targeted RNAi mediated gene silencing in the cattle tick Rhipicephalus (Boophilus) microplus
