At 25 d of age, 1 piglet closed to the average weight was randomly chosen from each litter, resulting in 4 piglets per treatment. Animals were then anesthetized by intramuscular injection of sodium pentobarbital (50 mg/kg BW) and then euthanized. Jejunal samples, approximately 2 cm in length, were resected from the middle portion of the jejunum, washed in PBS, and frozen in liquid N2 and stored at - 80°C before used for the isolation of total RNA.
Growth protocol
A total of fourty 21-d old pigs (Duroc × Landrace) from 4 litters (10 piglets per litter) were assigned randomly to control (suckling) and weaning groups on the basis of their litter origins (n = 20/ group). Piglets in control group continued to be nursed by sows, whereas piglets in weaning group were weaned and moved from the farrowing pens to nursery pens without mixing any litters (5 piglets per pen), and had ad libitum access to the basal diet and water with 14.48 MJ/kg DE and 20.50% CP (N × 6.25).
Extracted molecule
total RNA
Extraction protocol
Total RNA of jejunum tissues was extracted following the Trizol Reagent instructions (Invitrogen), and checked for a RNA integrity number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies).Qualified total RNA was further purified by RNeasy mini kit (Qiagen) and RNase-Free DNase Set (Qiagen). Only those samples that had an OD260/OD280 ratio of approximately 2.0 and showed no degradation (RNA integrity number ≥ 7.0) were used to generate labeled targets.
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit (Agilent technologies), following the manufacturer’s instructions. Cy3 was used to label the samples from the control sucking individuals and the weaned animals.
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions
Scan protocol
Slides were scanned by Agilent Microarray Scanner (G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit.
Description
Gene expression in 25d old weaned piglet's jejunum Weaned-rep2
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
