8 protocols
AccessionNameType
P-MTAB-27363
labeling
Agilent Two-Color Microarray-Based Gene Expression (Quick Amp Labeling)
Version: 5.7
Agilent publication number: G4140-90050
URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_Two-Color_GE_5.7.pdf
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
P-MTAB-27362
pool
A equal aliquot of all Cy3 respective Cy5 labeled extracts was pooled to build a reference.
P-MTAB-27366
bioassay_data_transformation
Data was log2 transformed. Data was background substracted and LOESS normalized using R (2.15)/Bioconductor/Limma
P-MTAB-27365
image_acquisition
Slides were scanned using an Agilent DNA Microarray Scanner G2505C; scan software: Agilent Scan Control Version A.8.1.3; quantification software: Agilent Feature Extraction Version 10.5.1.1 (FE Protocol GE_105_Dec08).(Parameters: Scanning hardware = G2505A DNA microarray scanner [Agilent], Scanning software = Feature Extraction Software [Agilent])
P-MTAB-27364
hybridization
Agilent Two-Color Microarray-Based Gene Expression (Hybridization)
Version: 5.7
Agilent publication number: G4140-90050
URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_Two-Color_GE_5.7.pdf
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
P-MTAB-27359
grow
HeLa cells were obtained from ATCC and cultured in DMEM (PAA, Coelbe, Germany) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 microg/ml streptomycin in a humidified incubator at 37 degrees C and 5% CO2.
P-MTAB-27360
compound_based_treatment
Cells were seeded at a density of 8*10^5 cells per 6 well in 2 ml DMEM with 10% FCS and cultured for 2h. 1280 ng siRNA in 100 microliter OptiMEM (Invitrogen) and 20 microliter HiPerfect (Qiagen, Hilden, Germany) were mixed and incubated for 5-10 min at room temperature prior to transfection. The cells were replated 24 h post-transfection at a density of 8*10^5 cells per 6 cm dish. Transfection was repeated 48 h after start the experiment, and cells were passaged after another 24 h. Twenty-four hours following the last transfection, cells were incubated in serum-free medium overnight. Cells were stimulated with 10 ng/ml IL-1beta and harvested after another 1 h.

Control siRNA (Dharmacon)
CAGUCGCGUUUGCGACUGG

siPPARD was a pool of siRNAs (Qiagen) with the following sequences:
Hs_PPARD_2: CAGACUGACGAAACUUUAATT
Hs_PPARD_3: GUGAUAUCAUUGAGCCUAATT
Hs_PPARD_5: GCGGAUCAAGAAGACCGAATT
Hs_PPARD_6: GGUUACCCUUCUCAAGUAUTT

si-HSP27 (HSPB1) was a pool of siRNAs (Dharmacon, siGENOME 4 Upgrade) with the following sequences:
si-HSP27_1: CAAGUUUCCUCCUCCCUGU
si-HSP27_3: CCGGAGGAGUGGUCGCAGU
siHSP27_4: GGCAGGACGAGCAUGGCUA
siHSP27_5: CCGAUGAGACUGCCGCCAA
P-MTAB-27361
nucleic_acid_extraction
RNA was isolated using the Nucleospin RNA II kit (Macherey-Nagel, Dueren, Germany) according to manufacturers instructions.