challenge: S. aureus cell type: White blood cells time: 30h after challenge sample type: White blood cells 30h after challenge
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with Trizol reagent following the instructions of the supplier (Invitrogen, Milan, Italy). High-quality RNA was obtained through purification with RNeasy MinElute spin column (Qiagen, Milan, Italy) and eluted in RNase-free water. RNA was then quantified using a NanoDrop spectrophotometer (Agilent, Santa Clara, CA) and quality-checked using a Bioanalyser 2100 (Agilent, Santa Clara, CA).
Label
Cy5
Label protocol
Total RNA (1μg) was used as a template to synthesize antisense RNA (aRNA) with Cy5-ULS using the RNA Amplification and Labelling Kit from CombiMatrix (ampULSe Cat. no. GEA-022; Kreatech Biotechnology, Amsterdam, The Netherlands) according to manufacturer's instructions
Hybridization protocol
Prehybridization was performed by incubating the arrays with prehybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 5x Denhardt's solution, 100 ng/µl Salmon Sperm DNA, 0.05% SDS) for 30 minutes at 45 °C. 4 µg of labeled aRNA were fragmented by incubation with 5 µl of Fragmentation Solution (200mM Tris Acetate pH 8.1, 500 mM KOAc, 150 mM MgOAc) for 20 min at 95 °C. Hybridization was performed at 45°C for 16 hours in hybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 25% DiFormamide, 100 ng/µl Salmon Sperm DNA, 0.04% SDS). Hybridization washings were performed as the following: - wash with 6X SSPET wash solution (6X SSPE, 0.05% Tween-20). - wash with 3X SSPET wash solution (3X SSPE, 0.05% Tween-20). - wash with 0.5X SSPET wash solution (0.5X SSPE, 0.05% Tween-20). - wash with PBST wash solution (2X PBS, 0.1% Tween-20). - 2 washes with 2X PBS
Scan protocol
After hybridization and washing, the microarray was dipped in imaging solution, covered with LifterSlip™, and then scanned using a GenePix 4000B microarray scanner (Axon, Toronto, CA) and the accompanying acquisition software (CombiMatrix Microarray Imager Software). Multiple scanning at different PMT was provided for each hybridization
Description
Goat6 _30h_blood
Data processing
Data were extracted and loaded into R software using the Limma analysis package from Bioconductor and the signal intensities were processed and normalized using standard procedures. Limma performs a linear regression analysis on the hybridizations, using a group-means parameterization approach to compare the different conditions and performs a false discovery rate adjustment with Benjamini-Hochberg correction for multiple testing [Smyth, 2004]. DE genes were selected using an adjusted P-value cut off equal to 0.01
