|
| Status |
Public on Feb 09, 2020 |
| Title |
pregnant (Days 15-16) vs. cyclic (Days 15-16 of the estrous cycle) 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Anterior pituitary of pregnant pigs
|
| Organism |
Sus scrofa domesticus |
| Characteristics |
reproductive status: Early pregnancy
race: Large White x Polish Landrace
days: days 15 to 16
days: days 15 to 16
|
| Growth protocol |
Eight mature gilts (Large White x Polish Landrace, weighing 90-110 kg) were used in the study (n = 8). All individuals were descended from private breeding farm. Day 0 was a day indicating the beginning of estrus. Cyclic animals (n=4) were sacrificed on days 15-16. Days of the estrous cycle were confirmed on the morphology of ovaries. Gilts assigned to the early pregnancy group (n = 4) were naturally bred twice on the first and the second days of the estrous and sacrificed on days 15-16 of pregnancy. Pregnancy was confirmed by the presence of conceptuses. Animals were slaughtered by qualified abattoir worker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from porcine pituitaries using the RNeasy Mini Kit (Qiagen, USA), according to manufacture procedure.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 7.5 and 10. The obtained nucleic acid was 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 pituitary of pregnant and cyclic females 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 the microarray manufacturer’s protocol, cRNA was fragmented and mixed with hybridization buffer.
|
| |
|
| Channel 2 |
| Source name |
Anterior pituitary of cyclic pigs
|
| Organism |
Sus scrofa domesticus |
| Characteristics |
reproductive status: The estrous cycle
race: Large White x Polish Landrace
|
| Growth protocol |
Eight mature gilts (Large White x Polish Landrace, weighing 90-110 kg) were used in the study (n = 8). All individuals were descended from private breeding farm. Day 0 was a day indicating the beginning of estrus. Cyclic animals (n=4) were sacrificed on days 15-16. Days of the estrous cycle were confirmed on the morphology of ovaries. Gilts assigned to the early pregnancy group (n = 4) were naturally bred twice on the first and the second days of the estrous and sacrificed on days 15-16 of pregnancy. Pregnancy was confirmed by the presence of conceptuses. Animals were slaughtered by qualified abattoir worker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from porcine pituitaries using the RNeasy Mini Kit (Qiagen, USA), according to manufacture procedure.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 7.5 and 10. The obtained nucleic acid was 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 pituitary of pregnant and cyclic females 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 the microarray manufacturer’s protocol, cRNA was fragmented and mixed with hybridization buffer.
|
| |
|
| |
| Hybridization protocol |
Two differentially labelled samples (from pregnant and cyclic gilts) 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' 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 pregnant and cyclic pigs.
|
| |
|
| Submission date |
Feb 08, 2020 |
| Last update date |
Feb 09, 2020 |
| Contact name |
Agata Zmijewska |
| E-mail(s) |
[email protected]
|
| Phone |
+48 89 5233201
|
| Organization name |
Univeristy of Warmia and Mazury
|
| Department |
Demartment of Animal Anatomy and Physiology
|
| Street address |
Oczapowski 1a
|
| City |
Olsztyn |
| ZIP/Postal code |
10-719 |
| Country |
Poland |
| |
|
| Platform ID |
GPL16571 |
| Series (1) |
| GSE144981 |
Transcriptomic analysis of the porcine anterior pituitary gland during the peri-implantation period |
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