Cows received one of the following treatments: weekly intramuscular injections (5 mL) of 1) saline 0.9% NaCl (B9˗B12˗); 2) 320 mg of folic acid (B9+B12-; pteroylmonoglutamic acid, MP Biomedicals, Solon, OH, USA); 3) 10 mg of vitamin B12 (cyanocobalamin, 5,000 µg/mL, Vetoquinol, Lavaltrie, Quebec, Canada); or 4) 320 mg of folic acid and 10 mg of vitamin B12.
Growth protocol
Mammary gland and hepatic tissues were obtained from the lactating dairy cows, 64 ± 3 days after calving.
Extracted molecule
total RNA
Extraction protocol
To obtain biological material, biopsies of the mammary gland and liver were performed using a rotating stainless steel cannula with a retractable blade at the cutting edge. The process of hepatic biopsies uses ultrasound guidance to avoid vascular ducts. Total RNA was extracted from hepatic and mammary tissues by using a QIAzol Lysis Reagent (QIAGEN Inc., Toronto, ON, Canada) following the original manufacturer’s protocol, with slight modifications. Briefly, frozen samples (100 mg of tissue) were homogenized in 2 mL of QIAzol Lysis Reagent on ice using a Tissue-Tearor. A volume of 600 µL QIAzol Lysis Reagent was added to 400 µL of homogenate; the mixture was vigorously vortexed and kept at room temperature for 5 min to promote dissociation of nucleoprotein complexes. A volume of 200 µL of chloroform was added; the mixture was shaken and left at room temperature for 3 min followed by a centrifugation at 12 000 × g for 15 min at 4°C to remove lipids. After centrifugation, the aqueous fraction (upper layer) was taken and RNA was precipitated by adding an equal volume of 70 % ethanol. RNA was purified according to manufacturer’s procedure using RNeasy Mini Kit (QIAGEN Inc., Toronto, ON, Canada), including on-column DNase digestion.
Label
Cy3
Label protocol
RNA was quantified using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, Inc.). Cyanine 3-labeled CTP cRNA was produced with 50ng of total RNA using the Low Input Quick Amp Labeling Kit, according to manufacturer’s instructions (Agilent Technologies, Inc). The quality of complimentary RNA (cRNA) was evaluated by capillary electrophoresis on NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1. The labeled cRNA was then normalized at 1.65µg (4-pack microarray format) and fragmented (Incubate at 60°C for exactly 30 minutes to fragment RNA and reaction stoped with Hi-RPM Hybridization Buffer. The cRNA yields and specific activities were as recommended.
Hybridization protocol
Samples were hybridized on Agilent expression Bovine (V2) 4x44k using the Agilent Gene Expression Hybridization Kit accoding to the manufacturer's instrutions (Agilent Technologies, Inc). The arrays were incubated in an Agilent Hybridization oven at 65°C for 17 hours at 10 rpm. They were washed and scanned on an Agilent DNA Microarray Scanner C. All these steps were done according to Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, Inc). 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 Agilent Technologies Scanner G2505C US09473739 (Grid name 023647_D_F_20110614) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%.
Description
Bovine GE Microarray 4x44K
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 023647_D_F_20110614) to obtain background and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Raw microarray expression intensities were corrected for background using normexp using the Saddle-point model parameter, according to Ritchie et al (Ritchie, Silver et al. 2007) and using an offset value of 16. The flag spots were replaced with blanks. Between-array normalization using quartile normalization parameter of the LOWESS was performed on the background corrected arrays. Normalized data are log transformed.
