|
Status |
Public on Dec 23, 2007 |
Title |
cow_control_biological rep3 |
Sample type |
RNA |
|
|
Source name |
biopsy of mammary parenchyma, milked twice daily, approx. day 7 of lactation
|
Organism |
Bos taurus |
Characteristics |
Breed: Holstein, multiparous cow
|
Treatment protocol |
Multiparous Holstein dairy cattle were milked either 2x or 4x daily during the first 21 days of lactation. Milking 4x daily was initiated at day 4 of lactation. Prior to day 4, all cows were milked 2x daily. Mammary biopsies were performed at approximately day 7 of treatment for extraction of total RNA and subsequent transcript profiling.
|
Growth protocol |
Multiparous Holstein dairy cattle were evaluated during the first week of lactation.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using the RNeasy Midi Kit (Qiagen, Valenica, CA). To remove contaminating genomic DNA, on-column DNase digestion was performed using the RNase-free DNase Set (Qiagen) according to kit instructions. RNA quality was assessed using the BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA) and concentrations were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE).
|
Label |
biotin
|
Label protocol |
Target was prepared according to the Affymetrix Technical Manual. Total RNA (2 μg) was converted to cDNA using reverse transcriptase (Invitrogen Corp., Carlsbad, CA) and a modified oligo(dT)24 primer that contains T7 promoter sequences (GenSet, San Diego, CA). After first strand synthesis, residual RNA was degraded by addition of RNaseH and a double-stranded cDNA molecule was generated using DNA polymerase I and DNA ligase. The cDNA was then purified and concentrated using a phenol:chloroform extraction, followed by ethanol precipitation. The cDNA products were incubated with T7 RNA polymerase and biotinylated ribonucleotides using the GeneChip IVT Labeling Kit (Affymetrix). The resultant cRNA product was purified using an RNeasy column (Qiagen) and quantified with a spectrophotometer.
|
|
|
Hybridization protocol |
The cRNA target (20 μg) was incubated at 94ºC for 35 min in fragmentation buffer (Tris, MgOAc, KOAc). The fragmented cRNA was diluted in hybridization buffer (MES, NaCl, EDTA, Tween 20, Herring Sperm DNA, Acetylated BSA) containing biotin-labeled OligoB2 and Eukaryotic Hybridization Controls (Affymetrix). The hybridization cocktail was denatured at 99°C for 5 min, incubated at 45°C for 5 min, and then injected into a GeneChip cartridge. The GeneChip array was incubated at 42°C for at least 16 h in a rotating oven at 60 rpm. GeneChips were washed with a series of nonstringent (25°C) and stringent (50°C) solutions containing variable amounts of MES, Tween20 and SSPE. The microarrays were then stained with streptavidin phycoerythrin and the fluorescent signal was amplified using a biotinylated antibody solution.
|
Scan protocol |
Fluorescent images were detected in a GeneChip Scanner 3000.
|
Description |
Gene expression data from bovine mammary gland under differential milking frequency
|
Data processing |
Expression data were extracted using the GeneChip Operating System v 1.2 (Affymetrix). An estimate of signal for each transcript was calculated using the Position-Dependent Nearest-Neighbor method (Zhang et al., 2003) with a compression adjustment, and the results were analyzed via principal components analysis (PCA). Based on the PCA results, the IMF4-4 sample was removed prior to comparing the groups. Raw fold-change for each transcript was calculated by taking the simple ratio of the geometric means of the signal values for each respective group. Differential expression was determined using a robust implementation of permutation testing (http://www.expressionanalysis.com/pdf/PADE_technote.pdf).
|
|
|
Submission date |
Oct 17, 2007 |
Last update date |
Aug 14, 2011 |
Contact name |
Erin E Connor |
E-mail(s) |
erin.connor@ars.usda.gov
|
Phone |
301-504-6104
|
Fax |
301-504-8414
|
Organization name |
USDA-ARS, BARC
|
Department |
Animal and Natural Resources Institute
|
Lab |
Bovine Functional Genomics Laboratory
|
Street address |
10300 Baltimore Avenue
|
City |
Beltsville |
State/province |
MD |
ZIP/Postal code |
20705 |
Country |
USA |
|
|
Platform ID |
GPL2112 |
Series (1) |
GSE9344 |
Gene expression in bovine mammary gland in response to increased milking frequency as determined by microarray and SAGE |
|