Treatment protocol
Exposure of cell cultures to hypoxia (1% oxygen) was undertaken in an In vivo2 Hypoxia Work Station (Ruskin Technologies) in parallel with cells maintained in normoxic conditions (21% oxygen). Cells were recovered after 16h, 32h and 48h of exposure to hypoxia.
Growth protocol
MCF-7 cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 2mmol/L of L-glutamine
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from cells using the miRVana miRNA isolation kit (Ambion) in accordance with the prescribed protocol provided with the kit. RNA quality was assessed after extraction using Agilent bioanalyser.
Label
Label protocol
100µg of total RNA were first dephosphorylated using Calf Instestine Alkaline Phosphatase (New England Biolabs). Single molecules of Cyanine 3-pCp provided in the miRNA Labeling Reagent and Hybridization kit (Agilent) were ligated to the 3' end of the dephosphorylated RNA molecules using T4 RNA ligase (New England Biolabs). Labeled RNAs were purified throughout micro Bio-Spin 6 columns (BioRad) and dried down using a speed-vacuum concentrator Savant ISS110 (Thermo Scientific). Samples were resuspended in 18µl of RNAse free water and prepared for hybridization by adding 4.5µl of 10X GE Blocking agent and 22.5µl of 2X Hi-RPM Hybridization Buffer (both in Agilent miRNA Labeling Reagent and hybridization kit).
Hybridization protocol
The hybridization was performed at 55˚C for 20h and at a constant speed rotation of 20rpm.
Scan protocol
After washing the microarrays with the Agilent Gene Expression Wash Buffer kit, the hybridization signals were detected by the Agilent Microarray scanner (G2565BA)and using the following settings: scan area 61 x 21.6 mm, scan resolution 5µm, 5µm scanning mode Single Pass, eXtended Dynamic range selected, Dye chanel set to green, green PMT set to XDR Hi 100% and XDR Lo 5%.
Description
Total RNA containing short RNA fraction
Hypoxia_32h_R2
Data processing
The scanned images were analyzed with Feature Extraction Software version 9.5.1 (Agilent). A GeneView file was generated for each of the samples and used for further analysis. The Total Gene signal from GeneView files was normalised using the vsn package from Bioconductor
Series (2)
Total number of rows:
821
