| Metadata |
| datasetIdentifier | PASS00597 |
| datasetType | MSMS |
| submitter | Hegang Li <lihegang2000@aliyun.com> |
| submitter_organization | Qingdao Institute of Animal Science and Veterinary Medicine |
| lab_head_full_name | Jinshan Zhao |
| lab_head_email | zhaojinshande@sohu.com |
| lab_head_organization | Qingdao Institute of Animal Science and Veterinary Medicine |
| lab_head_country | China |
| datasetTag | Aohanskinfollicle |
| datasetTitle | Identification of skin-expressed genes possibly associated with wool growth regulation of Aohan fine wool sheep |
| publicReleaseDate | 2014-10-22 00:00:00 |
| finalizedDate | 2014-10-22 18:32:27 |
| summary | Background: Sheep are valuable resources for the animal fibre industry. Therefore, identifying genes which regulate wool growth would offer strategies for improving the quality of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side (hair-rich) and groin (hairless) skins of Aohan fine wool sheep (a Chinese indigenous breed).
Results: Comparing the body side to the groin skins (S/G) of Aohan fine wool sheep, the microarray study revealed that 1494 probes were differentially expressed, including 602 more highly expressed and 892 less highly expressed probes. The microarray results were verified by means of quantitative PCR. Cluster analysis could distinguish the body side skin and the groin skin. Based on the Database for Annotation, Visualization and Integrated Discovery (DAVID), 38 of the differentially expressed genes were classified into four categories, namely regulation of receptor binding, multicellular organismal process, protein binding and macromolecular complex. Proteomic study revealed that 187 protein spots showed significant (p < 0.05) differences in their respective expression levels. Among them, 46 protein entries were further identified by MALDI-TOF/MS analyses.
Conclusions: Microarray analysis revealed thousands of differentially expressed genes, many of which were possibly associated with wool growth. Several potential gene families might participate in hair growth regulation. Proteomic analysis also indentified hundreds of differentially expressed proteins.
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| contributors | Nan Liu, Hegang Li, Kaidong Liu, Juanjuan Yu, Ran Bu, Ming Cheng, Wei De, Jifeng Liu, Guangling He, Jinshan Zhao |
| publication | Nan Liu, Hegang Li, Kaidong Liu, Juanjuan Yu, Ran Bu, Ming Cheng, Wei De, Jifeng Liu, Guangling He, Jinshan Zhao.BMC Genetics,submitted |
| growth | These three sheep were reared normally in pens. |
| treatment | All animals were treated in accordance with the animal protocols defined by national and local animal welfare bodies, and all animal work was approved by the Shandong Province Biological Studies Animal Care and Use Committee.One ram and two ewes of 16-month-old Aohan fine wool sheep were used in the microarray study. These animals were half sibs (sharing the same father). In December 2010, two areas of full-thickness skin were sampled from the same animal under local anaesthesia: body side skin (wool bearing) and groin skin (non-wool bearing) for microarray and proteomic experiments. The area of each sample was about 1 cm2. |
| extraction | Lysis buffer preparation: 42% Urea, 15.2% Thiourea, 4% CHAPS, 1% DTT. Sampled tissues were homogenziated in lysis buffer (containing 1% cocktail and 2% IPG-buffer, added right before use) at the ratio of 1:7 (weight/volume). The tissues were cut into small pieces by ophthalmic scissors, and left at 4℃ for 1h, vortexed it every 15 min. Then, the tissue homogenate was centrifuge at 40,000g for 30 min. Supernatants were collected and stored at -80℃. Protein concerntrations were determined by Bradford method. |
| separation | 1.One-dimensional electrophoresis
0.5% IPG-buffer was added into each 150 μg protein sample (in a final volume of 400-600μL), and was loaded in the One-dimensional electrophoresis instrument. The progamme is as follows: Step-n-hold (S1, 30V for 6h; S2, 60V for 6h); Gradient (S3, 500V for 1h;S4,1000V for 1h; S5, 3000V for 3h; S6, 8000V for 3h); Step-n-hold (S7, 8000V for 20h).
2.2-dimensional (2-D) SDS-PAGE preparation
Tris-HCl (PH=8.8), Monomer storage (30% Acrylamid and 0.8% NN’-methy lenebisacry lamid), 10×electrophoresis buffer (3.03% Tris-Base, 14.4% Glycine, 1% SDS), balanced solution (36.05% Urea, 5% Tris-HCl, 2% SDS, 34.5% Glycerine).
3.The second dimensional SDS-PAGE
The electrophoresis programme is as follows:
Transfer: Voltage 300v, Current 50mA, Time 1h.
Separation: Voltage 300v, Current 200~250mA, Time 4~5h.
Fixative preparation: 40% Ethanol and 10% Acetic acid.
Electrophoresis was carried out until the blue dye front had just disappeared from the bottom of the gel.
Fixation: take out the rubber strip and put it into Fixative for 1h.
4.Staining and visualization
Sensitizing solution preparation: 30% Ethanol, 0.314% Na2S2O3, 6.8%NaAc.
Sensitizing: the gels were sensitized for 30 min.
Washing: the gels were washed for three times using ddH2O. Ten minutes each time.
Ilver staining: Silver staining solution was prepared (1.25 AgNO3 and 200μL Formaldehyde in 500mL ddH2O). The gels were stained by the solution for 20 min.
Washing: the gels were washed for 2 min using ddH2O.
Visualizing solution preparation: 12.5g Na2CO3 and 100μL Formaldehyde in 500mL ddH2O.
Termination solution preparation: 2g Glycine in 50ml ddH2O.
Visualization until the solution became muddy, then terminating for 30min. |
| digestion | |
| acquisition | |
| informatics | Determination of relative protein expression
Gels were then scanned and analyzed using ImageMaster TM 2D platinum software (Version 5.0, GE Healthcare, San Francisco, CA, USA). The expression level was determined by the relative volume of each spot in the gel and expressed as %Vol (%Vol = [spot volume / Σvolumes of all spots resolved in the gel]). The means and standard deviations of both sample groups were calculated. Statistical significance with Student’s t-tests using ImageMaster TM 2D platinum software. P values﹤0.05 were considered statistically significant. |
| instruments | Ultraflex II MALDI-TOF-TOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) |
| species | Sheep |
| massModifications | None |
