|
| Status |
Public on Nov 09, 2018 |
| Title |
Adiponectin treated vs non-treated 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pituitary 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 10 to 12
tissue: Anterior pituitary lobes
cell type: pituitary cells
treatment: none (untreated control)
|
| Treatment protocol |
Anterior pituitary lobes were washed with fresh DMEM, minced into 1- to 2-mm pieces and trypsinised with 0.25% trypsin (7–8 times, 10 min., 37°C). Dispersed pituitary cells were transferred to plastic tubes, repeatedly centrifuged at 1,200 g for 10 min, and washed with DMEM three times. After the final centrifugation, pituitary cells were filtered through a nylon filter (60 μm mesh) and counted in a hemocytometer. Cell viability (90–97%) was determined by trypan blue dye exclusion. Pituitary cells were then resuspended in McCoy’s 5A medium with 10% horse serum, 2.5% foetal calf serum (FCS), 240 U/ml nystatin and 100 μg/ml gentamycin, supplemented with 0.1% vitamins and 0.01% nonessential amino acids. One millilitre of dispersed cell suspension (1 x 10^6/ml) was transferred to each 24-well culture plate. The cells were allowed to attach for 48 h (37°C, 95% O2 and 5% CO2). After preculture, the cells were incubated for 24 h (37°C, 95% O2 and 5% CO2) in 1ml of McCoy’s 5A medium (without serum) containing bacitracin (2 x 10-5 M) with or without (control culture) recombinant human adiponectin (BioVendor, USA) at a concentration of 10 μg/ml.
|
| Growth protocol |
The study was performed on mature gilts (Large White × Polish Landrace) aged 7 to 8 months, with body weights of 120–130 kg, raised on a private farm, as described in our previous study. Pituitaries (n=4) were harvested in the mid-luteal phase (day 10-12) of the porcine oestrous cycle. Gilts were observed daily for oestrus behaviour in the presence of an intact boar. The onset of the second oestrus was marked as day 0 of the oestrous cycle. The mid-luteal phase of the oestrous cycle was also confirmed based on ovarian morphology. Pigs were slaughtered by qualified abattoir worker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from each AP cell culture was isolated with the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. Additionally, RNA was purified by on-column digestion of DNA with DNase I (Qiagen, USA). The concentration and purity of the isolated RNA were determined by spectrophotometry (Infinite 200 PRO, Tecan Group, Switzerland). The RNA integrity number (RIN) was evaluated in the Agilent Bioanalyser 2100 (Agilent Technology, USA). RNAs were stored at −80 °C until the microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
The RNA isolated from the control and adiponectin treated cells 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 |
pituitary adiponectin-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 10 to 12
tissue: Anterior pituitary lobes
cell type: pituitary cells
treatment: recombinant human adiponectin (10 μg/ml)
|
| Treatment protocol |
Anterior pituitary lobes were washed with fresh DMEM, minced into 1- to 2-mm pieces and trypsinised with 0.25% trypsin (7–8 times, 10 min., 37°C). Dispersed pituitary cells were transferred to plastic tubes, repeatedly centrifuged at 1,200 g for 10 min, and washed with DMEM three times. After the final centrifugation, pituitary cells were filtered through a nylon filter (60 μm mesh) and counted in a hemocytometer. Cell viability (90–97%) was determined by trypan blue dye exclusion. Pituitary cells were then resuspended in McCoy’s 5A medium with 10% horse serum, 2.5% foetal calf serum (FCS), 240 U/ml nystatin and 100 μg/ml gentamycin, supplemented with 0.1% vitamins and 0.01% nonessential amino acids. One millilitre of dispersed cell suspension (1 x 10^6/ml) was transferred to each 24-well culture plate. The cells were allowed to attach for 48 h (37°C, 95% O2 and 5% CO2). After preculture, the cells were incubated for 24 h (37°C, 95% O2 and 5% CO2) in 1ml of McCoy’s 5A medium (without serum) containing bacitracin (2 x 10-5 M) with or without (control culture) recombinant human adiponectin (BioVendor, USA) at a concentration of 10 μg/ml.
|
| Growth protocol |
The study was performed on mature gilts (Large White × Polish Landrace) aged 7 to 8 months, with body weights of 120–130 kg, raised on a private farm, as described in our previous study. Pituitaries (n=4) were harvested in the mid-luteal phase (day 10-12) of the porcine oestrous cycle. Gilts were observed daily for oestrus behaviour in the presence of an intact boar. The onset of the second oestrus was marked as day 0 of the oestrous cycle. The mid-luteal phase of the oestrous cycle was also confirmed based on ovarian morphology. Pigs were slaughtered by qualified abattoir worker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from each AP cell culture was isolated with the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. Additionally, RNA was purified by on-column digestion of DNA with DNase I (Qiagen, USA). The concentration and purity of the isolated RNA were determined by spectrophotometry (Infinite 200 PRO, Tecan Group, Switzerland). The RNA integrity number (RIN) was evaluated in the Agilent Bioanalyser 2100 (Agilent Technology, USA). RNAs were stored at −80 °C until the microarray analysis.
|
| Label |
Cy5
|
| Label protocol |
The RNA isolated from the control and adiponectin treated cells 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 adiponectin 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_252644010751_S01_GE2_107_Sep09_1_3.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 adiponectin.
|
| |
|
| 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) |
| GSE122311 |
The transcriptomic profile of porcine anterior pituitary cells is influenced by adiponectin |
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