For acute systemic candidiasis, mice in groups of eight to ten were challenged by intravenous tail-vein injection of an inoculum of 1 x 10E7 (lethal infection group) and 1 x 10E5 c.f.u. (sublethal infection group), respectively, in a 200 µl of a Candida tropicalis suspension via a 25-gauge syringe. In control group, 100 µl of PBS was used instead of the yeast suspension.
Growth protocol
The end-point of infection evaluation was day 7 in this model. At the time of killing, mice were anesthetized and exsanguinated by cardiac puncture. Whole blood and serum was collected and stored in -80 °C for future usage.
Extracted molecule
total RNA
Extraction protocol
RNA isolation was performed by using Mouse RiboPure™-Blood RNA Isolation Kit (Ambion Inc, USA) according to manufacturer’s instructions. Briefly, the frozen whole blood was thawed at room temperature and centrifuged at high speed to separate the blood cells from RNAlater. Lysis solution was added to the blood pellet and the cells were vortexed vigorously. Subsequently, samples were extracted with sodium acetate and acid-phenol chloroform, centrifuged and precipitated with 0.5 volume of 100 % ethanol (Merck KGaA, USA). Then, samples were further purified by using filter cartridge and eluted with nuclease free water. DNase digestion was carried out by utilizing TURBO DNAfree™ (Ambion Inc, USA). In addition, GLOBINclear™ (Ambion Inc, USA) was used to deplete alpha and beta globin mRNA from total RNA preparations by employing the biotin/ streptavidin binding, nucleic acid hybridization and magnetic bead separation. Briefly, the globin mRNA was captured by using biotinylated oligonucleotide mix and hybridized to streptavidin magnetic beads. Then, the bound globin mRNA with streptavidin magnetic beads were removed by magnetic field, producing RNA with depleted globin mRNA. Finally, the RNA was purified using rapid magnetic bead-based purification method and eluted with nuclease free water.
Label
Cy3
Label protocol
Illumina TotalPrep RNA amplification (Ambion Inc, USA) was used in cDNA synthesis and in vitro transcription. Briefly, the first-strand cDNA was synthesized from 100 ng of total RNA with T7 Oligo (dT) primer which contained T7 promoter sequence. The second strand cDNA synthesis was then synthesized in second-strand Master Mix for 2 h at 16 °C. Then, cDNA was purified with filter cartridge and eluted with nuclease free water. All enzymes were from Ambion (Ambion Inc, USA). Following purification, in vitro transcription from the cDNA sample was carried out using T7 enzyme mix (Ambion Inc, USA) to generate biotin-labeled cRNA. Subsequently, the labeled cRNA was separated from unincorporated ribonucleotides by passing through filter cartridge.
Hybridization protocol
The cRNA hybridization mix was heated to 65 °C for 5 min and equilibrated to room temperature. Aliquots of each sample (750 ng of cRNA in 15 µl of the hybridization mix) were hybridized to the corresponding bead arrays, inserted in BeadChip Hyb Chamber (Illumina, USA) and incubated at 58 °C for 16 h in a rocker speed Hyb Oven (Illumina, USA) set a speed of 5. Then, the arrays were washed with 1 X High Temperature Wash Buffer (Illumina, USA), E1BC Buffer (Illumina, USA), blocked with E1 Buffer (Illumina, USA), stained with streptavidin-Cy3 (GE Healthcare, USA) and washed again. The washing procedures were carried out in Hybex Water Bath (Illumina, USA) and orbital shaker. Lastly, the arrays were dried by low-speed centrifugation and scanned with Illumina BeadStation (Illumina, USA).
Scan protocol
Arrays were dried by low-speed centrifugation and scanned with Illumina BeadStation (Illumina, USA). Scanning were performed according to manufacturer's suggestion.
Description
Six-week-old female BALB/c mice (weighing 20-25g) were used for all animal experiments. The animals were randomized, assigned to groups of eight to ten individuals and given food and water ad libitum.
Data processing
Raw data from BeadStudio Version 3.0 (Illumina, USA) was imported to Genespring 7.0 (Agilent, USA) for preprocessing and data analysis. The data for each array were normalized accordingly to per-chip normalization to the distribution of all genes on that array as well as the median expression for that gene across all chips in the study and data transformation. The different sets of data in the mice groups were compared with each other by one-way analysis of variance (ANOVA) test with a P-value cut-off of 0.05.
