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Sample GSM2178543 Query DataSets for GSM2178543
Status Public on Jun 06, 2016
Title [652_-7 / 5_75 cy5]-[9277_-7 / 4_125 cy3]
Sample type RNA
 
Channel 1
Source name Liver Biopsy
Organism Bos taurus
Characteristics tissue: Liver
bcs: 5
feeding (%): 75
sampling time (days from parturition): -7
Treatment protocol Approximately 200 days before calving, cows were allocated randomly to one of six treatment groups in a 2 x 3 factorial arrangement: two pre-calving BCS categories (4.0 and 5.0; based on a 10-point scale: BCS4 and BCS5, respectively) and three levels of energy intake during the three wk preceding calving (75, 100, and 125% of estimated requirements). Cows in the BCS4 and BCS5 groups were managed through late lactation to ensure target calving BCS was achieved at dry off. Cows were then maintained until three wk before expected calving date, at which point they were managed within their pre-allotted pre-calving energy intake treatments by offering different allowances of fresh pasture/cow per day.
Growth protocol Grazing dairy cows were managed through the dry period, untill 30 days post partum
Extracted molecule total RNA
Extraction protocol Liver tissue (30-50 mg) was homogenized in a TissueLyser II (Qiagen GmbH) for 2 × 2 min bursts at 30 Htz. Total RNA and DNA were extracted using a Qiagen AllPrep DNA/RNA mini kit (Qiagen GmbH) as per manufacturer’s instructions and RNA was treated with DNase (Ambion DNA-free kit; Ambion Inc., Austin, TX). The RNA quantity, purity, and integrity were determined using a NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE) and the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA). Average RNA Integrity Number (RIN) for samples at -7, 7, and 28 d relative to parturition were 8.18 (± 0.39), 7.77 (± 0.43), and 8.17 (± 0.45), respectively. RNA samples were stored at -80 °C until analysis.
Label cy5
Label protocol The Agilent platform was chosen to conduct the microarray experiment, using the 44K Bovine (V2) gene expression microarray chip (Agilent Technologies Inc.), following the manufacturer’s protocols. Briefly, a total of 200 ng of RNA per sample were used to generate first-strand cDNA, which was reverse transcribed to cRNA using the low-input quick amp labeling kit (Agilent Technologies Inc). The resulting cRNA was labeled with either Cy3 or Cy5 fluorescent dye, purified using RNeasy mini spin columns (Qiagen), and subsequently eluted in 30 μL of DNase-RNase-free water. The NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA) was used to confirm the manufacturer’s recommended criteria for yield and specific activity of at least 0.825 μg and ≥6.
 
Channel 2
Source name Liver Biopsy
Organism Bos taurus
Characteristics tissue: Liver
bcs: 4
feeding (%): 125
sampling time (days from parturition): -7
Treatment protocol Approximately 200 days before calving, cows were allocated randomly to one of six treatment groups in a 2 x 3 factorial arrangement: two pre-calving BCS categories (4.0 and 5.0; based on a 10-point scale: BCS4 and BCS5, respectively) and three levels of energy intake during the three wk preceding calving (75, 100, and 125% of estimated requirements). Cows in the BCS4 and BCS5 groups were managed through late lactation to ensure target calving BCS was achieved at dry off. Cows were then maintained until three wk before expected calving date, at which point they were managed within their pre-allotted pre-calving energy intake treatments by offering different allowances of fresh pasture/cow per day.
Growth protocol Grazing dairy cows were managed through the dry period, untill 30 days post partum
Extracted molecule total RNA
Extraction protocol Liver tissue (30-50 mg) was homogenized in a TissueLyser II (Qiagen GmbH) for 2 × 2 min bursts at 30 Htz. Total RNA and DNA were extracted using a Qiagen AllPrep DNA/RNA mini kit (Qiagen GmbH) as per manufacturer’s instructions and RNA was treated with DNase (Ambion DNA-free kit; Ambion Inc., Austin, TX). The RNA quantity, purity, and integrity were determined using a NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE) and the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA). Average RNA Integrity Number (RIN) for samples at -7, 7, and 28 d relative to parturition were 8.18 (± 0.39), 7.77 (± 0.43), and 8.17 (± 0.45), respectively. RNA samples were stored at -80 °C until analysis.
Label cy3
Label protocol The Agilent platform was chosen to conduct the microarray experiment, using the 44K Bovine (V2) gene expression microarray chip (Agilent Technologies Inc.), following the manufacturer’s protocols. Briefly, a total of 200 ng of RNA per sample were used to generate first-strand cDNA, which was reverse transcribed to cRNA using the low-input quick amp labeling kit (Agilent Technologies Inc). The resulting cRNA was labeled with either Cy3 or Cy5 fluorescent dye, purified using RNeasy mini spin columns (Qiagen), and subsequently eluted in 30 μL of DNase-RNase-free water. The NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA) was used to confirm the manufacturer’s recommended criteria for yield and specific activity of at least 0.825 μg and ≥6.
 
 
Hybridization protocol The labeled cRNA was fragmented and then hybridized to the microarray slide according to manufacturer’s protocol. Briefly, 825 ng of Cy3 and Cy5 labeled cRNA sample were combined, mixed with 11 μL of 10× Blocking Agent (Agilent Technologies Inc.), 2.2 μL of 25× Fragmentation Buffer (Agilent Technologies Inc.), and nuclease-free water (to a final volume of 55 μL); and then fragmented at 60°C for 30 s. The reaction was then stopped by adding 55 μL of 2× GEx Hybridization Buffer (Agilent Technologies Inc.), and the samples were loaded onto the slide. These were hybridized in a rotating hybridization oven (Agilent Technologies Inc.) at 65°C for 17 h.
Scan protocol The slides were washed according to the manufacturer’s recommended procedures and scanned using a GenePix 4000B scanner (Axon Instruments Inc., Sunnyvale, CA) and GenePix Pro v.6.1 software. Resulting spots where features were substandard were flagged as bad and excluded from subsequent analysis.
Description Sample were total RNA extracted from bovine (American Holstein) liver biopsy tissue
Data processing Array quality was assessed using an in-house parser written in Perl language. Spots that received a -100 flag by GenePix 6.0 were removed from further analysis and background intensity was subtracted from the foreground intensity. Spots on the slide were considered “good” if the median intensity was ≥3 standard deviation above median background for each channel (i.e., dye). Spots were flagged “present” when both dyes passed the criteria, “marginal” if only one dye passed the criteria, or “absent” when both dyes failed to pass the criteria. Statistical analysis was conducted on oligos that were flagged as “present” and “marginal”. Data from a total of 30 microarrays were normalized for dye and array effects (i.e., Loess normalization and array centering and scaling) before statistical analysis.
 
Submission date May 26, 2016
Last update date Jun 07, 2016
Contact name Mario Pietro Emilio Vailati Riboni
Organization name University of Illinois at Urbana-Champaign
Department Animal Science
Street address 1207 W Gregory Dr
City Urbana
State/province IL
ZIP/Postal code 61801
Country USA
 
Platform ID GPL11648
Series (1)
GSE81958 Prepartum body condition score and plane of nutrition affect the hepatic transcriptome during the transition period in grazing dairy cows

Data table header descriptions
ID_REF
VALUE lowess normalized log2 ratio (Cy3/Cy5)

Data table
ID_REF VALUE
5550
29836 0.471305719
12438
39508
37833 -0.256339753
11301 -0.30256277
10065
3417 -1
16918 0.596367264
23390 -0.199609026
42974 0.192645078
5828 0.84424233
6676 0.004224586
23092
14158 1.649092838
44573 -0.167158567
10482 0.48673204
21186
13576 0.319894698
16618 0.700439718

Total number of rows: 43803

Table truncated, full table size 644 Kbytes.




Supplementary file Size Download File type/resource
GSM2178543_4A_-_2015-03-10.gpr.gz 6.0 Mb (ftp)(http) GPR
Processed data included within Sample table
Processed data are available on Series record

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