|
| Status |
Public on Nov 14, 2018 |
| Title |
OXB treated vs non-treated 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
myometrium non-treated
|
| Organism |
Sus scrofa domesticus |
| Characteristics |
treatment: non-treated
period of pregnancy: days 15 to 16
race: Large White x Polish Landrace
gilts age and weight: mature/7-8 months/120 to 130 kg
|
| Treatment protocol |
The myometrial explants obtained from the middle of uterine horns were cut into small, irregular slices (3 mm of thickness, 100 mg ± 10%). Tissue explants were washed three times in medium M199. The tissue explants were placed in the separate sterile culture vials with 2 mL medium 199 containing. Cultures were preincubated for 2 h (37°C, 95% O2, 5% CO2). To determine the influence of OXB on the global gene expression in the myometrium, slices were treated with OXB at the concentration of 10 nM and incubated for another 24 h at the same conditions. Control tissues were incubated without any treatment. All cultures were prepared in duplicates in four separate experiments for each group (n = 4). The myometrial tissue explants were frozen and stored at -80°C for further analyses.
|
| Growth protocol |
Four mature gilts (Large White x Polish Landrace) were used in the study (n = 4). All individuals were descended from private breeding farm. The gilts were at the age of 7-8 months and the weight of 120 to 130 kg. All animals used in the study were on days 15 to 16 of gestation. The day after coitus was marked as the first day of gestation. Insemination was performed on days 1 to 2 of the oestrous cycle. Pregnancy was confirmed by the presence of conceptuses. 15 to 16 days after coition, gilts were sacrificed. Pigs 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 OXB 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 OXB-treated
|
| Organism |
Sus scrofa domesticus |
| Characteristics |
gilts age and weight: mature/7-8 months/120 to 130 kg
treatment: myometrium OXB-treated
period of pregnancy: days 15 to 16
race: Large White x Polish Landrace
|
| Treatment protocol |
The myometrial explants obtained from the middle of uterine horns were cut into small, irregular slices (3 mm of thickness, 100 mg ± 10%). Tissue explants were washed three times in medium M199. The tissue explants were placed in the separate sterile culture vials with 2 mL medium 199 containing. Cultures were preincubated for 2 h (37°C, 95% O2, 5% CO2). To determine the influence of OXB on the global gene expression in the myometrium, slices were treated with OXB at the concentration of 10 nM and incubated for another 24 h at the same conditions. Control tissues were incubated without any treatment. All cultures were prepared in duplicates in four separate experiments for each group (n = 4). The myometrial tissue explants were frozen and stored at -80°C for further analyses.
|
| Growth protocol |
Four mature gilts (Large White x Polish Landrace) were used in the study (n = 4). All individuals were descended from private breeding farm. The gilts were at the age of 7-8 months and the weight of 120 to 130 kg. All animals used in the study were on days 15 to 16 of gestation. The day after coitus was marked as the first day of gestation. Insemination was performed on days 1 to 2 of the oestrous cycle. Pregnancy was confirmed by the presence of conceptuses. 15 to 16 days after coition, gilts were sacrificed. Pigs 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 OXB 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 OXB 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.
|
| 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 OXB.
|
| |
|
| Submission date |
Nov 11, 2018 |
| Last update date |
Nov 15, 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) |
| GSE122414 |
The in vitro influence of orexin B on the porcine myometrium on days 15 to 16 of gestation. |
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