|
| Status |
Public on May 20, 2015 |
| Title |
HUVEC treated with IL-1 replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HUVEC Untreated
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Umbilical Vein Endothelial Cells (HUVEC)
treaetd with: none (untreated control)
|
| Treatment protocol |
Three condition experiment: DHA treated HUVEC cells (25 µmol/L DHA for 48 hours) vs control; IL-1 treated HUVEC cells (5ng/ml for 3 hours) vs control; HUVEC treated with 25 µmol/L DHA for 48 hours and then stimulated with 5 ng/mL IL-1β for 3 hours. For each condition, 3 replicates
|
| Growth protocol |
Human umbilical vein endothelial cells (HUVEC) were isolated from segments of umbilical cords from normal-term deliveries, and cultured in Medium 199
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Milan, Italy) according to manufacturer’s instructions. RNA quality and integrity were assessed using the Nanodrop spectrophotometer (Nanodrop Technologies, Montchanin, U.S.A) and 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) respectively. Samples with ratios of absorbance at 280/260 nm between 1.8 and 2.0 and at 280/230 nm between 2.0 and 2.2, and with RNA integrity number (RIN) of ≥ 9 were judged to be of acceptable quality and integrity to be used for microarray labelling.
|
| Label |
Cy5
|
| Label protocol |
300ng of total RNA per each condition was amplified and labeled in the presence of cyanine 3-CTP and cyanine 5-CTP using the Agilent Low RNA Input Linear Amp Kit following the manufacturer's protocol. Dye-incorporation rate and cRNA output were quantified with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Channel 2 |
| Source name |
HUVEC treated with IL-1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Umbilical Vein Endothelial Cells (HUVEC)
treated with: 5ng/ml iL-1 for 3 hrs
|
| Treatment protocol |
Three condition experiment: DHA treated HUVEC cells (25 µmol/L DHA for 48 hours) vs control; IL-1 treated HUVEC cells (5ng/ml for 3 hours) vs control; HUVEC treated with 25 µmol/L DHA for 48 hours and then stimulated with 5 ng/mL IL-1β for 3 hours. For each condition, 3 replicates
|
| Growth protocol |
Human umbilical vein endothelial cells (HUVEC) were isolated from segments of umbilical cords from normal-term deliveries, and cultured in Medium 199
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Milan, Italy) according to manufacturer’s instructions. RNA quality and integrity were assessed using the Nanodrop spectrophotometer (Nanodrop Technologies, Montchanin, U.S.A) and 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) respectively. Samples with ratios of absorbance at 280/260 nm between 1.8 and 2.0 and at 280/230 nm between 2.0 and 2.2, and with RNA integrity number (RIN) of ≥ 9 were judged to be of acceptable quality and integrity to be used for microarray labelling.
|
| Label |
Cy3
|
| Label protocol |
300ng of total RNA per each condition was amplified and labeled in the presence of cyanine 3-CTP and cyanine 5-CTP using the Agilent Low RNA Input Linear Amp Kit following the manufacturer's protocol. Dye-incorporation rate and cRNA output were quantified with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| |
| Hybridization protocol |
The hybridization procedure was conducted according to the Two-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Tecnologies ). Briefly, 825ng of the correspondiing Cy3- and Cy5-labeled cRNA were combined and hybridized overnight (17 hours at 65°C) to Agilent Whole Human Genome Oligo Microarrays 4x44K (G4112F, Part No. G4112-60520; Batch No. 0006060953) 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 air dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using two color scan setting for 4x44k array slides. Microarray slides were scanned at 5 uM resolution using an extended dynamic range (XDR)
|
| Description |
IL-1.2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE12_107_Sep09 and Grid: 014850_D_F_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
May 20, 2014 |
| Last update date |
May 20, 2015 |
| Contact name |
Rosanna Martinelli |
| E-mail(s) |
rmartinelli@unisa.it
|
| Phone |
+39 089965040
|
| Organization name |
Salerno University
|
| Department |
Medicine and Surgery
|
| Street address |
Via Allende
|
| City |
Baronissi (SA) |
| ZIP/Postal code |
84081 |
| Country |
Italy |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE57825 |
DHA effect on endothelial cells (HUVEC) under proinflammatory condition |
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