|
| Status |
Public on Nov 09, 2018 |
| Title |
OXA treated vs non-treated myometrium 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
myometrium non-treated
|
| Organism |
Sus scrofa domesticus |
| Characteristics |
race: Large White x Polish Landrace
gilts age: mature; 7-8 months
gilts weight: 120 to 130 kg
period of pregnancy: days 15 to 16
tissue: myometrial tissue obtained from the middle of uterine horns
treatment: none (untreated control)
|
| Treatment protocol |
Small, irregular explants (3 mm of thickness, 100 mg ± 10%) of the myometrial tissue were obtained from the middle of uterine horns. Tissue explants were washed three times in medium M199 and placed in the separate sterile culture vials with 2 mL medium 199 containing 0.1% BSA , 5% dextran/charcoal-stripped new-born calf serum. Explants were preincubated for 2 h (37°C, 95% O2, 5% CO2). To examine the influence of OXA on the myometrial transcriptome, slices were treated with OXA (10 nM) and incubated for another 24 h at the same conditions. Control tissues were incubated in the absence of OXA, under the same conditions. All cultures were prepared in duplicates in four separate experiments for each group (n = 4). After the incubation, myometrial explants were frozen and stored at -80°C for further analyses.
|
| Growth protocol |
Four mature gilts descended from the private breeding farm (Large White x Polish Landrace) were used in the study (n = 4). All animals were at the age of 7-8 months and the weight of 120 to 130 kg. The gilts used in the study were on days 15 to 16 of gestation. As the first day of gestation we considered the first day after coition. Gilts were inseminated on days 1 to 2 of the oestrous cycle. The presence and morphology of conceptuses confirmed the stage of pregnancy. 15 to 16 days after coition, gilts were slaughtered by qualified abattoir worker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from tissues using peqGOLD TriFast isolation system according to the manufacturer’s instructions. RNA purity and quantity were measured spectrophotometrically . RNA quality was checked using RNA 6000 Nano Assay Kit on an Agilent 2100 Bioanalyzer. RNA integrity number (RIN) score between 8 and 10 were used for microarray analysis and quantitative real-time PCR (qPCR) experiments. RNAs were stored at −80 °C until the microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
The RNA isolated from the control and OXA treated tissues were labelled with Cyanine-3 (Cy-3) and Cyanine-5 (Cy-5), respectively. Quantity and purity of obtained cRNA were measured with NanoQuant plates on the Infinite M200 Pro plate reader . 825 ng of Cy-3 and 825 ng of Cy-5-labeled cRNA were mixed together (for each microarray). According to microarray manufacturer’s protocol, cRNA was fragmented and mixed with hybridization buffer.
|
| |
|
| Channel 2 |
| Source name |
myometrium OXA-treated
|
| Organism |
Sus scrofa domesticus |
| Characteristics |
race: Large White x Polish Landrace
gilts age: mature; 7-8 months
gilts weight: 120 to 130 kg
period of pregnancy: days 15 to 16
tissue: myometrial tissue obtained from the middle of uterine horns
treatment: OXA (10 nM)
|
| Treatment protocol |
Small, irregular explants (3 mm of thickness, 100 mg ± 10%) of the myometrial tissue were obtained from the middle of uterine horns. Tissue explants were washed three times in medium M199 and placed in the separate sterile culture vials with 2 mL medium 199 containing 0.1% BSA , 5% dextran/charcoal-stripped new-born calf serum. Explants were preincubated for 2 h (37°C, 95% O2, 5% CO2). To examine the influence of OXA on the myometrial transcriptome, slices were treated with OXA (10 nM) and incubated for another 24 h at the same conditions. Control tissues were incubated in the absence of OXA, under the same conditions. All cultures were prepared in duplicates in four separate experiments for each group (n = 4). After the incubation, myometrial explants were frozen and stored at -80°C for further analyses.
|
| Growth protocol |
Four mature gilts descended from the private breeding farm (Large White x Polish Landrace) were used in the study (n = 4). All animals were at the age of 7-8 months and the weight of 120 to 130 kg. The gilts used in the study were on days 15 to 16 of gestation. As the first day of gestation we considered the first day after coition. Gilts were inseminated on days 1 to 2 of the oestrous cycle. The presence and morphology of conceptuses confirmed the stage of pregnancy. 15 to 16 days after coition, gilts were slaughtered by qualified abattoir worker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from tissues using peqGOLD TriFast isolation system according to the manufacturer’s instructions. RNA purity and quantity were measured spectrophotometrically . RNA quality was checked using RNA 6000 Nano Assay Kit on an Agilent 2100 Bioanalyzer. RNA integrity number (RIN) score between 8 and 10 were used for microarray analysis and quantitative real-time PCR (qPCR) experiments. RNAs were stored at −80 °C until the microarray analysis.
|
| Label |
Cy5
|
| Label protocol |
The RNA isolated from the control and OXA treated tissues were labelled with Cyanine-3 (Cy-3) and Cyanine-5 (Cy-5), respectively. Quantity and purity of obtained cRNA were measured with NanoQuant plates on the Infinite M200 Pro plate reader . 825 ng of Cy-3 and 825 ng of Cy-5-labeled cRNA were mixed together (for each microarray). According to microarray manufacturer’s protocol, cRNA was fragmented and mixed with hybridization buffer.
|
| |
|
| |
| Hybridization protocol |
Two differentially labelled samples (from OXA treated and non-treated, control samples) were placed on each array (one slide, n = 4) in a balanced block design with dye swaps. Hybridization was carried out in an Agilent hybridization oven for 17 h at 60°C. After the hybridization process, the microarrays were washed.
|
| Scan protocol |
Microarrays were scanned at 5 μm resolution with an Agilent’s High-Resolution C Microarray Scanner according to the manufacturers's protocol.
|
| Description |
US45103052_252644011042_S01_GE2_107_Sep09_1_1.txt:cy5ByCy3Signal
|
| Data processing |
The TIFF image, generated after the scanning process, was used for: data extraction and detailed analysis, background subtraction from features, and dye normalizations (linear and LOWESS); in the Feature Extraction Software. Gene expression data were analysed in GeneSpring GX 12 software (Agilent Technologies, USA) to identify the genes that were differentially expressed in pigs that were and were not treated with OXA.
|
| |
|
| Submission date |
Nov 08, 2018 |
| Last update date |
Nov 09, 2018 |
| Contact name |
Kamil Dobrzyn |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Warmia and Mazury
|
| Department |
Department of Animal Anatomy and Physiology
|
| Lab |
Faculty of Biology and Biotechnology
|
| Street address |
Oczapowskiego, 1A
|
| City |
Olsztyn |
| State/province |
Warminsko-Mazurskie |
| ZIP/Postal code |
10 719 |
| Country |
Poland |
| |
|
| Platform ID |
GPL16571 |
| Series (1) |
| GSE122310 |
The in vitro effect of orexin A on the porcine myometrial transcriptomic profile during early-implantation period |
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