isolated cells: PMNL dietary treatment: M days from parturition: -14
Extracted molecule
total RNA
Extraction protocol
PMNL: Coccygeal venous or arterial blood samples (~120 mL) were collected in vacutainer tubes containing acid citrate dextrose (ACD Solution A; Fisher Scientific) on days -14 (±2) and +7 relative to parturition (~0700 h). Samples were placed on ice until PMNL isolation, which took place within 30 min of collection. PMNL were isolated from whole blood by sample centrifugation, cell lysing, and several rounds of centrifugation with PBS washing. Isolated PMNL were homogenized at full speed in a solution of 2 mL TRIzol reagent (Invitrogen, Carlsbad, CA) with 1 µL linear acrylamide (Ambion, Inc., Austin, TX). Homogenate was stored at -80ºC in RNA-free microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA) for further analysis. RNA: Once samples were thawed and centrifuged, total RNA was separated with chloroform and acid phenol:chloroform (Ambion, Inc., Austin, TX). Total RNA was precipitated with isopropanol and cleaned with 75% ethanol. Resuspension in RNA storage buffer (Ambion, Inc., Austin, TX) allowed RNA to be stored at -80ºC. Prior to storage, RNA purity was confirmed using a NanoDrop ND-1000 (NanoDrop Technologies, Rockland, DE) OD260mm/OD280mm ratio, and RNA quality was recorded using a 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) RNA integrity number (RIN). Average RIN was 7.7 ± 0.2.
Label
Cy3
Label protocol
The microarray platform for the present study used 4x44k-Agilent Bovine (V2) Gene Expression Microarray chips (Agilent Technologies; cat# G2519F-023647). Labeling and hybridization protocols were followed as directed by Agilent Technologies. cDNA was created from 200 ng total quantity of RNA, then reverse-transcribed into Cy-3 or Cy-5 fluorescent dye-labeled cRNA using a Low-Input Quick Amp Labeling Kit (two colors; Agilent Technologies; cat# 5190-2306). Resulting cRNA was purified using RNeasy mini spin columns (Qiagen; cat# 74104), and eluted in nuclease-free water. Yield of at least 825 ng and specific activity ≥ 6.0 pmol/µg were confirmed using the NanoDrop ND-1000 (NanoDrop Technologies). Samples were stored at -80ºC until hybridization.
isolated cells: PMNL dietary treatment: S days from parturition: -14
Extracted molecule
total RNA
Extraction protocol
PMNL: Coccygeal venous or arterial blood samples (~120 mL) were collected in vacutainer tubes containing acid citrate dextrose (ACD Solution A; Fisher Scientific) on days -14 (±2) and +7 relative to parturition (~0700 h). Samples were placed on ice until PMNL isolation, which took place within 30 min of collection. PMNL were isolated from whole blood by sample centrifugation, cell lysing, and several rounds of centrifugation with PBS washing. Isolated PMNL were homogenized at full speed in a solution of 2 mL TRIzol reagent (Invitrogen, Carlsbad, CA) with 1 µL linear acrylamide (Ambion, Inc., Austin, TX). Homogenate was stored at -80ºC in RNA-free microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA) for further analysis. RNA: Once samples were thawed and centrifuged, total RNA was separated with chloroform and acid phenol:chloroform (Ambion, Inc., Austin, TX). Total RNA was precipitated with isopropanol and cleaned with 75% ethanol. Resuspension in RNA storage buffer (Ambion, Inc., Austin, TX) allowed RNA to be stored at -80ºC. Prior to storage, RNA purity was confirmed using a NanoDrop ND-1000 (NanoDrop Technologies, Rockland, DE) OD260mm/OD280mm ratio, and RNA quality was recorded using a 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) RNA integrity number (RIN). Average RIN was 7.7 ± 0.2.
Label
Cy5
Label protocol
The microarray platform for the present study used 4x44k-Agilent Bovine (V2) Gene Expression Microarray chips (Agilent Technologies; cat# G2519F-023647). Labeling and hybridization protocols were followed as directed by Agilent Technologies. cDNA was created from 200 ng total quantity of RNA, then reverse-transcribed into Cy-3 or Cy-5 fluorescent dye-labeled cRNA using a Low-Input Quick Amp Labeling Kit (two colors; Agilent Technologies; cat# 5190-2306). Resulting cRNA was purified using RNeasy mini spin columns (Qiagen; cat# 74104), and eluted in nuclease-free water. Yield of at least 825 ng and specific activity ≥ 6.0 pmol/µg were confirmed using the NanoDrop ND-1000 (NanoDrop Technologies). Samples were stored at -80ºC until hybridization.
Hybridization protocol
After thawing cRNA, 825 ng of one Cy-3-labeled sample and 825 ng of one Cy-5-labeled sample were mixed together and with 11 µL of 10X Blocking Agent (Agilent Technologies; cat# 5188-5281) and 2.2 µL of 25X Fragmentation Buffer (Agilent Technologies; cat# 5185-5974). Nuclease-free water was added to bring the final volume of solution to 55 µL. Solution was placed in a 60ºC water bath for 30 min, then 55 µL of 2X GEx Hybridization Buffer (Agilent Technologies; cat# 5190-0403) was added to stop fragmentation reactions. The total solution of 110 µL was loaded onto a Hybridization Gasket Slide (Agilent Technologies; cat# G2534-60011), and hybridized to a microarray slide for 17 h in a 65ºC hybridization oven.
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) blood PMNL animal protocol: 8 cows were assigned to one of two groups which differed in dietary energy levels fed throughout the 45-d dry period. One group received at least 100% calculated NEL (CON; 1.34 Mcal/kg DM) from a diet high in wheat straw while the other received over 140% calculated NEL (OVE; 1.62 Mcal/kg DM) from a corn silage-based diet. Both diets were fed once daily (0600 h) using an individual gate feeding system (American Calan, Northwood, NH, USA). All cows were housed in a ventilated, enclosed barn for the entire dry period. After calving, cows were fed a common lactation diet (NEL = 1.69 Mcal/kg DM) as TMR once daily (0600 h) and housed in a tie-stall barn. Milking occurred twice daily (0400 and 1600 h).
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.
