|
| Status |
Public on Nov 24, 2015 |
| Title |
WholebloodAnalysis_Untreated_60day_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood, Holstein Friesian, lactating, Untreated,60 day, replicate 3
|
| Organism |
Bos taurus |
| Characteristics |
breed: Holstein Friesian
tissue: whole blood
gender: female
|
| Treatment protocol |
Initial (0 days) and end of study (60 day) blood samples were drawn from the jugular vein aseptically from the Probiotics-treated animals (n=5) and control animals (n=5) into ACD tubes. The blood samples were incubated for 30 minutes at 37oC with 5% CO2. The Fastrack Microbial (Probiotics) Pack (Conklin Company, Kansas City, MO) Ingredients: Yeast culture (Saccharomyces cerevisiae), rice hulls, calcium carbonate, dried chicory root, dried Enterococcus faecium fermentation product, dried Lactobacillus acidophilus fermentation product, dried Aspergillus oryzae, fermentation extract, dried Bacillus subtilis fermentation extract. Recommended Dose for Lactating dairy cattle: Supplement at the rate of 1 to ounces per animal daily.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted using the ZR whole blood RNA miniprep kit (ZYMO RESEARCH, Irvine, CA) following the manufacturer's recommendations. The protocol includes differential lysis of red and white blood cells, and an on-column DNase digestion. Total RNA was quantified using a NanoDrop-1000 spectrophotometer and RNA integrity and quality was determined with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples with high RNA integrity (RIN>7) were stored at -80oC until used for microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug (RIN>7) pooled RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNA 1.63 ug (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Bovine v2 4×44k Oligo Microarrays (G2519F) 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 DNA Microarray Scanner (G2505B) using one color scan default settings for 4x44k array slides.
|
| Description |
Gene expression of end of study (60day) cow blood sample of untreated control animals receiving only water (Control)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (GE1-v5_95_Feb07) to obtain background subtracted and spatially detrended Processed Signal intensities. Data normalization and statistical analysis were performed using GeneSpring software 13.0 (Agilent). Fold changes in gene expression, t-test, gene ontology and pathway analysis were generated using the GeneSpring software.
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| |
|
| Submission date |
Nov 23, 2015 |
| Last update date |
Nov 24, 2015 |
| Contact name |
Mulumebet Worku |
| Organization name |
North Carolina Agricultural and Technical State University
|
| Department |
Animal Sciences
|
| Lab |
Genomic diversity and animal biotechnology
|
| Street address |
1601 E market Street
|
| City |
Greensboro |
| State/province |
North Carolina |
| ZIP/Postal code |
27411 |
| Country |
USA |
| |
|
| Platform ID |
GPL11649 |
| Series (1) |
| GSE75240 |
Global gene expression profiling of the effect of probiotics supplementation in dairy cows |
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