|
| Status |
Public on Jan 04, 2021 |
| Title |
EX_H_44P |
| Sample type |
RNA |
| |
|
| Source name |
porcine endometrial tissue
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: endometrium
gender: female
age: 8 months
|
| Treatment protocol |
Randomly selected horns within each gilt received hormonal infusions: PGE2 (200 µg/infusion) simultaneously with E2 (33.3 µg/infusion). The contralateral horn received placebo infusions. In the control group each horn received intrauterine placebo infusions. Treatments were administered every 4 h for 24 h on days 11-12 after the onset of estrus.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from endometrial samples using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Purity and concentration of isolated RNA was measured with NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA integrity was assessed by using Agilent Eukaryote Total RNA Nano chips and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
|
| Label |
CY3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 mL containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to gilent 4x44k Porcine Gene Expression microarrays (G2519F-026440) for 17 hours at 65 °C in a rotating Agilent hybridization oven. 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 (G2505C) using one color scan setting for Agilent 4x44k Porcine Gene Expression microarrays (G2519F-026440).
|
| Description |
Endometrial sample collected from gilt assigned to the experimental group (hormone-treated)
Gene expression after 24h of treatment
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Expression signals were filtered based on “well above background” flags (detection in 5 of 10 samples in control group, and in 5 of 6 samples in experimental group) and normalized with the BioConductor package VSN (Huber et al. 2002).
|
| |
|
| Submission date |
Sep 25, 2020 |
| Last update date |
Jan 05, 2021 |
| Contact name |
Piotr Kaczynski |
| E-mail(s) |
[email protected]
|
| Phone |
+48 89 539 31 80
|
| Organization name |
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences
|
| Department |
Department of Hormonal Action Mechanisms
|
| Street address |
Tuwima 10
|
| City |
Olsztyn |
| ZIP/Postal code |
10-748 |
| Country |
Poland |
| |
|
| Platform ID |
GPL16571 |
| Series (1) |
| GSE158568 |
Synergistic action of estradiol and PGE2 on endometrial transcriptome in vivo resembles pregnancy effects better than estradiol alone |
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