Pig age:gestation day 90; Pig tissue: fetal placenta; Erhualian pig
Extracted molecule
total RNA
Extraction protocol
placentas from Erhualian gestation day75 and 90 or Large White gestation day 75 and 90 all were snap frozen in liquid nitrogen after collection and stored at –80℃ until RNA extraction. TRIzol (Invitrogen) and RNeasy Mini Kit (QIAGEN) were used for RNA extractions and purifications respectively followed by manufacturer’s instructions. RNA quality and concentration were checked by Agilent Bioanalyser 2100.
Label
biotin
Label protocol
GeneChip® IVT Labeling Kit (Affymetrix) protocol: 1.Transfer the needed amount of template cDNA (12 μL) to RNase-free microfuge tubes and add Rnase-free Water(8 μL), 10× IVT Labeling Buffer (4 μL), IVT Labeling NTP Mix(12 μL), IVT Labeling Enzyme Mix(4 μL) in the order. 2. Carefully mix the reagents and collect the mixture at the bottom of the tube by brief (5 second) microcentrifugation. 3. Incubate at 37°C for 16 hours. To prevent condensation that may result from water bath-style incubators,incubations are best performed in oven incubators for even temperature distribution, or in a thermal cycler. 4.Store labeled cRNA at -20°C, or -70°C if not purifying immediately.
Hybridization protocol
1.Mix the following components together(Hybridization solution). Fragmented cRNA: 5 μg; Control Oligonucleotide B2(3nM): 1.7 μL; 20× Eukaryotic Hybridization Controls(bioB, bioC, bioD, cre): 5 μL; Herring Sperm DNA(10mg/mL): 1 μL; Acetylated BSA(50mg/mL): 1 μL; 2× Hybridization Buffer: 50 μL; DMSO: 10μL; H2O: 31.3μL; 2. Equilibrate probe array to room temperature immediately before use. 3. Heat the hybridization cocktail to 99°C for 5 minutes in a heat block. 4. Meanwhile, wet the array by filling it through one of the septa with appropriate volume of 1X Hybridization Buffer using a micropipettor and appropriate tips. 5. Incubate the probe array filled with 1X Hybridization Buffer at 45°C for 10 minutes with rotation. 6. Transfer the hybridization cocktail that has been heated at 99°C, in step 3, to a 45°C heat block for 5 minutes. 7. Spin hybridization cocktail(s) at maximum speed in a microcentrifuge for 5 minutes to remove any insoluble material from the hybridization mixture. 8. Remove the buffer solution from the probe array cartridge and fill with appropriate volume of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube. 9. Place probe array into the Hybridization Oven, set to 45°C. Avoid stress to the motor; load probe arrays in a balanced configuration around the axis. Rotate at 60 rpm. 10. Hybridize at 45℃ for 16 h with constant rotation at 60rpm. 11. After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of the following Non-Stringent Wash Buffer(Wash Buffer A:6X SSPE, 0.01% Tween-20). Post Hyb Wash #1: 10 cycles of 2 mixes/cycle with Wash Buffer A at 25°C. 12. Preparing the Staining Reagents. 12.1 SAPE Stain Solution(Mix well and divide into two aliquots of 600 μL each to be used for stains 1 and 3): 2X Stain Buffer 600.0 μL; 50 mg/mL BSA 48.0 μL; 1 mg/mL Streptavidin Phycoerythrin (SAPE) 12.0 μL; DI H20 540.0 μL; 12.2 Antibody Solution: 2X Stain Buffer 300.0 μL; 50 mg/mL BSA 24.0 μL; 10 mg/mL Goat IgG Stock 6.0 μL; 0.5 mg/mL biotinylated antibody 3.6 μL; DI H20 266.4 μL; 13. Post Hyb Wash #2: 4 cycles of 15 mixes/cycle with Wash Buffer B(100 mM MES, 0.1M [Na+], 0.01% Tween-20) at 50°C. 14. Stain: Stain the probe array for 10 minutes in SAPE solution at 25°C. 15. Post Stain Wash: 10 cycles of 4 mixes/cycle with Wash Buffer A at 25°C. 16. 2nd Stain: Stain the probe array for 10 minutes in antibody solution at 25°C. 17. 3rd Stain: Stain the probe array for 10 minutes in SAPE solution at 25°C. 18. Final Wash: 15 cycles of 4 mixes/cycle with Wash Buffer A at 30°C. The holding temperature is 25°C. Annotation: Chips were washed and stained with a GeneChip Fluidics Station 450 (Affymetrix).
Scan protocol
The probe arrays were scanned using the Affymetrix® GeneChip® Scanner 3000. Apply the spots just before scanning: 1. On the back of the probe array cartridge, clean excess fluid from around septa. 2. Carefully apply one Tough-Spots to each of the two septa. Press to ensure that the spots remain flat. If the Tough-Spots do not apply smoothly, that is, if you observe bumps, bubbles, tears, or curled edges, do not attempt to smooth out the spot. Remove the spot and apply a new spot. 3. Insert the cartridge into the scanner and test the autofocus to ensure that the Tough-Spots do not interfere with the focus. Scanning the Probe Array: 1. Select Run → Scanner from the menu bar. Alternatively, click the Start Scan icon in the tool bar. ⇒ The Scanner dialog box appears with a drop-down list of experiments that have not been run. 2. Select the experiment name that corresponds to the probe array to be scanned. A previously run experiment can also be selected by using the Include Scanned Experiments option box. After selecting this option, previously scanned experiments appear in the drop-down list. 3. By default, for the GeneArray® Scanner only, after selecting the experiment the number [2] is displayed in the Number of Scans box to perform the recommended 2X image scan. For the GeneChip® Scanner 3000, only one scan is required. 4. Once the experiment has been selected, click the Start button. ⇒ A dialog box prompts you to load an array into the scanner. 5. If you are using the GeneArray® Scanner, click the Options button to check for the correct pixel value and wavelength of the laser beam. ■ Pixel value = 3 μm ■ Wavelength = 570 nm If you are using the GeneChip Scanner 3000, pixel resolution and wavelength are preset and cannot be changed. 6. Open the sample door on the scanner and insert the probe array into the holder. Do not force the probe array into the holder. Close the sample door of the scanner. 7. Click OK in the Start Scanner dialog box. ⇒ The scanner begins scanning the probe array and acquiring data. When Scan in Progress is selected from the View menu, the probe array image appears on the screen as the scan progresses.
Description
No
Data processing
There are 11 paired perfect match (PM) and mismatch (MM) 25-mer probes in the Affymetrix GeneChip porcine genome array for each probeset representing a gene. The signals from these probe pairs are used to determine whether a given gene is expressed and to measure the gene expression level. The probe-pair (PM-MM) data were used to detect the expression level of genes on the array (present call, marginal call, and absent call) by MAS 5.0 (Wilcoxon signed rank test). Raw data from .CEL files were converted to gene signal files by MAS 5.0 (Ver.2.3.1). Natural logarithms were then taken on quantile normalization（GeneSpring 7.31. Identification of DE genes was conducted using SAS (Ver.8.1, T-test). The p value cutoff for DE genes was set at 0.05.
