|
| Status |
Public on Aug 26, 2015 |
| Title |
HE_nodox_biorep2 |
| Sample type |
RNA |
| |
|
| Source name |
Hemogenic endothelium (HE) cells, noDox replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Hemogenic endothelium (HE) cells
treatment: without induction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction the cell pellet was resuspended in 800 µl Trizol™ and stored at -80ºC freezer until used. Before isolation, Trizol was thawed and 200µl chloroform was added to the Trizol™. Complementary DNA (cDNA) was prepared from the mRNAs using MMLV-RT (Promega M170A) as per manufacturers’ recommendations. Real time PCR was performed with SYBR Green PCR master mix (Life technologies, 4309155) in a Stepone qPCR machine.
|
| Label |
Cy3
|
| Label protocol |
RNA was labelled with Cyanine 3-CTP according to the sample preparation protocol from Agilent: One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling). The cells were vortexed and pelleted. The aqueous phase was collected and an equal volume of isopropanol was added and mixed. The mixture was loaded onto a RNeasy Minelute column (Qiagen, 74204) and purified as per manufacturer’s instructions. RNA concentration was determined by a nanodrop and its integrity was checked by Bioanalyzer.
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| |
|
| Hybridization protocol |
Hybridisation samples were prepared using 600 ng cRNA each according to the hybridisation protocol from Agilent: One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling), loaded onto the array slide and hybridised at 65 °C overnight
|
| Scan protocol |
After washing the microarray was scanned on an Agilent C Scanner using the Profile AgilentG3_GX_1Color for 8x60K microarrays (Dye channel: Green; Scan region: Scan Area (61 x 21.6 mm); Scan resolution (μm): 3; Tiff: 20 bit). The microarrays used were Agilent SurePrint G3 Mouse 8X60K microarrays (catalogue number: G4852A-028005).
|
| Description |
without induction
|
| Data processing |
The microarray gene expression scanned images were analysed with Feature Extraction Software 10.7.1.1 (Agilent) (protocol GE1_107_Sep09, Grid: 028005_D_F_20100614 and platform Agilent SurePrint G3 Mouse GE 8x60K). The raw data output by Feature Extraction Software was analysed using the LIMMA R package 37 with quantile normalisation and background correction by the using of normexp method 38 and an offset value of 16. Contrast matrix, lmFit and eBays function were used and a p value cut-off <= 0.001 was applied. Only genes with a minimum log2 intensity value equal to or greater than 6.5 in at least one array were selected as expressed genes. Genes that changed expression at least two fold up or down with respect to -/+ Dox or genes that changed expression at least two fold up or down through differentiation from haemogenic endothelium (HE) cells progressing to progenitors cells were considered as differentially expressed.
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| |
|
| Submission date |
Dec 29, 2014 |
| Last update date |
Aug 26, 2015 |
| Contact name |
Salam Adli Assi |
| E-mail(s) |
s.a.assi@bham.ac.uk
|
| Organization name |
University of Birmingham
|
| Department |
Institute for Cancer and Genomic Sciences
|
| Street address |
IBR
|
| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13912 |
| Series (2) |
| GSE64541 |
Cell stage dependent transcriptional response to leukemic oncogene |
| GSE64625 |
RUNX1/ETO role's in hematopoietic system |
|
