tissue: spleen gender: male treatment: Cordyceps militaris polypeptide at 800 mg/kg once daily continuously for 45 days
Treatment protocol
The experimental animals were fed under the conditions with a relative humidity of 40-70%, at 20±1℃ and in a sterile environment, with ad libitum access to water and food. After they adapted to the environment for one week, the mice were randomly divided into blank control group, model group, and low-, medium- and high-dose Cordyceps militaris polypeptide (CMP) groups. Mice in low-, medium- and high-dose Cordyceps militaris polypeptide were orally given 32, 160and 800 mg/kg once daily continuously for 45 days, respectively, and those in the blank control group and model group were given equal volume of distilled water in the same way. In addition to those in the blank control group, mice in the other groups were injected with 40 mg/kg of cyclophosphamide intraperitoneally once daily, successively for 2 day for the establishment of mice immunodeficiency model. 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.
Extracted molecule
total RNA
Extraction protocol
Briefly, the liver 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 45d 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.
