|
| Status |
Public on Oct 16, 2016 |
| Title |
252644010227_S01_GE1_107_Sep09_1_1 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood, pregnant, replicate 4
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: whole blood
gender: female
parity: fifth
|
| Treatment protocol |
Peripheral blood sample was collected at 14 days after insemination from the submandibular vein of each sow, thoroughly mixed with the cell lysate (Bioteke, Beijing, China) within 20 min, and stored at -70°C until further processing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from peripheral blood samples using blood total RNA fast extraction kit (Bioteke, Cat No. RP4001) and column purified using an RNeasy® Kit (QIAGEN, Cat No. 74104) according to the protocols. RNA quality was further assessed by calculating RIN (RNA integrity number) with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA samples that passed the quality control metrics, i.e. 2100 RIN >= 7.0 and 28S/18S > 0.7, were used for microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.05-0.2ug RNA using the One-Color Low Input Quick Amp Labeling Kit (Agilent technologies Santa Clara, US) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, GmBH, Germany). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 UV-VIS spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55ul of 2x GEx hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Agilent Porcine Oligo Microarray（4×44K）(design ID：026440）) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Microarray Scanner (G2565CA, Santa Clara, CA, US) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in pregnant sow blood
|
| Data processing |
The scanned images were analyzed with Feature Extraction software 10.7 (Agilent) using default parameters to obtain Raw data. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent).
|
| |
|
| Submission date |
Oct 02, 2014 |
| Last update date |
Oct 16, 2016 |
| Contact name |
Chuanli Zhou |
| E-mail(s) |
[email protected]
|
| Organization name |
China Agricultural University
|
| Department |
Department of Animal Breeding and Genetics
|
| Lab |
Key Laboratory of Animal Genetics and Breeding of Ministry of Agriculture
|
| Street address |
No.2 YuanmingyuanWest Road
|
| City |
Beijing |
| ZIP/Postal code |
100193 |
| Country |
China |
| |
|
| Platform ID |
GPL16571 |
| Series (1) |
| GSE61986 |
Differiential gene expression profiling in peripheral blood between pregnant sows versus non-pregnant sows |
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