Agilent One-Color Microarray-Based Gene Expression (Hybridization) Version: 5.7 Agilent publication number: G4140-90040 URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90040_One-Color_GE_5.7.pdf This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based one-color gene expression analysis.
array scanning protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (Agilent G2565AA and Agilent G2565BA) using one color scan setting for 4x44k array slides ( Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%). (Parameters: Scanning hardware = G2565BA DNA microarray scanner [Agilent], Scanning software = Feature Extraction Software [Agilent])
nucleic acid extraction protocol
Total RNA was isolated from frozen LD samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions and dissolved in RNase-free water. Total RNA concentration was assessed by spectrophotometry (OD 260 nm) and adjusted to a final concentration of 2.0ug/ul. The integrity and purity of the RNA were determined by the absorbance ratio at 260/280 nm and 2100 RIN. (Parameters: Extracted product = total_RNA, Amplification = none)
After birth,the longissimus dorsi (LD) samples were collected from the newborn piglets immediately, and washed briefly with PBS before being frozen in liquid nitrogen for detection.
In the FD group, standard pig feed without additional folate was provided to the pregnant sows from pregnancy.In the N group, the sows were fed standard pregnant sow feed (NRC 1998) with 1.3 mg/kg of added folate; this meets the folate requirement of pregnant sows. After 60 days of gestation, the sows were fed an ordinary feed designed for late pregnant sows. (Parameters: time unit = seconds, temperature unit = C)