240 Eight-week-old male ICR mice (SPF grade), weighing 18~22g, were purchased from Liaoning Changsheng Biotechnology Co., LTD (license number: SCXK (Liao)-2015-0001). Animals were housed individually in cages at 20±1℃and in 40-70% humidity, subjected to 12h light/dark cycle with free access to food and water.ICR mice were randomly divided into control group, Cy model group, Sch A low-dose, middle-dose, and high-dose group. Except those in the control group, mice in the other groups were given 40 mg/kg Cy in intraperitoneal injection for 5d for the establishment of immunodeficiency mice model. On the next day after the modeling, mice in low-, middle- and high-dose Sch A groups were intragastrically given 0.2g/kg/d, 0.4g/kg/d and 1g/kg/d Sch A respectively, and those in the control group and Cy model group were given an equal volume of distilled water in the same way successively for 4 weeks. Changes in the mice’s body weight and general status were regularly observed weekly. The mice’s body weights were measured every week regularly for the observation on changes in their body weights and their general states were also observed at the same time. we choose best group to do mRNA expression microarray.
Extracted molecule
total RNA
Extraction protocol
Briefly, the spleen tissue was ground into powders under the frozen condition with liquid nitrogen, and before the liquid nitrogen was volatilized, the liver powder was transferred into a 1.5ml microcentrifuge tube containing Trizol reagent (500mg of the liver tissue per 0.5 ml Trizol reagent). The liver powder in the microcentrifuge tube was drawn and re-injected vigorously several times with a 5 ml disposable syringe until the tissue homogenate was no longer sticky, in favor of the full breaking down of the cells and genomic DNA. The tissue homogenate was left to stand at room temperature for use. According to the ratio of 200ul chloroform per 1mlTrizol reagent, chloroform was added to the homogenate. The mixed solution was oscillated violently for 30sec for evenly mixing and then left to stand at room temperature for 5min. The mixed sample solution was centrifuged at 12000r/m for 15min to obtain the supernatant and the obtained supernatant was carefully transferred to another centrifuge tube, in which it was noticeable not to draw the intermediate phase. 500ul isopropanol was added to the supernatant, which was left to stand at the room temperature for 10min for its precipitation, and then centrifuged at 12000r/m for 15min and the supernatant was discarded to obtain the precipitation. 1Ml of 75% ethanol was added to the precipitation for the wash of RNA by oscillating it with a vortex, which was left to stand at room temperature for 5min and then centrifuged at 7500r/m for 5min to discard the supernatant. Finally, 75% ethanol was added to the precipitation to wash the RNA again, the precipitation solution was centrifuged at 7500r/m for 5min, the supernatant was removed, and the final precipitation was dried at room temperature, in which the RNA precipitation should not be too dried, otherwise it was not easy to dissolve.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 700μL RNA using the RNeasy Mini Kit (Qiagen p/n 74104) according to the manufacturer's instructions, followed by RNAeasy column purification. The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
Hybridization protocol
1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60°C for 30 min, and finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100 μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide.The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
Scan protocol
The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).
Description
Gene expression after 5d in mouse spleen
Data processing
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, genes that at least 3 out of 9 samples have flags in Detected (“All Targets Value”) were chosen for further data analysis. Differentially expressed genes with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed genes between the two samples were identified through Fold Change filtering. Hierarchical Clustering was performed using the R scripts. GO analysis and Pathway analysis were performed in the standard enrichment computation method.
