cell id: PE P passage #: 2 cell type: umbilical cord fibroblast diagnosis: preeclampsia
Treatment protocol
All cells for RNA collection were cultured in low glucose (5.6 mM)-DMEM medium containing 10% FBS, 1% NEAA, 2mM glutamine, 0.1mM beta-mercaptoethanol and 4ng/ml FGF2 under 4% or 20 O2 in 37 ℃ incubator. The human umbilical cord fibroblast cultures were maintained and expanded by media in every two days. When the culture reached 80-90% confluency, cells were detached dissociated with TrypLE and split at ratio of 1:4 into new T-25 flasks coated with 0.1% gelatin.
Growth protocol
Human umbilical cord (UC) tissues were washed at least twice with phosphate buffered saline (PBS) to remove blood cells and cut into two pieces. One was placed in high glucose (HG, 25 mM), the other in low glucose (LG, 5.6 mM) DMEM medium, respectively. Tissues were minced into 1–2 mm3 fragments with scissors and placed into a 48-well plate (one small piece per well, 12 wells for HG and 12 wells LG medium, respectively) that had been coated with 0.1% gelatin in HG/LG medium containing 10% FBS, 1% NEAA, 2mM glutamine, 0.1mM 2-mercaptoethanol and 4ng/ml FGF2, and cultured at 37C in an atmosphere of either 4 % O2/5 % CO2/91 % N2 or 5 % CO2/air (20 % O2) respectively to generate outgrowths of adherent cells.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer?s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).
Label
biotin
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Description
replicate 1
Data processing
The data were normalised using quantile normalisation
