|
| Status |
Public on Nov 21, 2015 |
| Title |
ExVivoBloodTreatment_CPE_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
BovinePeripheral Blood, CPE, replicate 4
|
| Organism |
Bos taurus |
| Characteristics |
breed: Holstein Friesian
tissue: whole blood
gender: Female
|
| Treatment protocol |
Freshly drawn peripheral blood from Holstein Friesian cows (n = 5) was treated with 10 ug/ml of cowpea leaf phenolic extract. Untreated samples served as negative control. The samples were incubated for 30 min at 37 ºC in a humidified incubator with 5% CO2.
|
| Growth protocol |
All experimental procedures were approved by the North Carolina A&T State University committee of animal care and use. Experimental animals were clinically healthy with no mastitis during the period of blood collection. Blood was collected from animals at the end of 60-day study of receiving FastTrack Microbial (Probiotics) pack (Conklin Company, Kansas City, MO).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the ZR whole blood RNA miniPrep kit (ZYMO RESEARCH, Irvine, CA) following the manufacturer's recommendations. The isolation 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 (Thermo Fisher Scientific, Waltham, MA) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). All samples displayed a 260/280 ratio greater than 1.8 and RNA integrity number (RIN) greater than 7.
|
| Label |
Cy3
|
| Label protocol |
All microarray procedures used were per manufacturer’s recommendation (Agilent Technologies, Santa Clara, CA). Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug 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 |
1.65 ug of Cy3-labelled cRNA (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 setting for 1x44k array slides.
|
| Description |
Gene expression after 30 min treatment of cow whole blood with 10ug of cowpea phenolic extract
|
| 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.
|
| |
|
| Submission date |
Nov 20, 2015 |
| Last update date |
Nov 21, 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) |
| GSE75239 |
Microarray analysis profiling of the effect of cowpea phenolic extract on gene expression in bovine peripheral blood in vitro |
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