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
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.
tissue: Liver bcs: 5 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
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.
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.
