|
| Status |
Public on Jun 30, 2013 |
| Title |
Carcinoma B4 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Pool of Synthetic microRNAs
|
| Organism |
synthetic construct |
| Characteristics |
reference: miRXplore Universal Reference provided by Miltenyi
|
| Extracted molecule |
other |
| Extraction protocol |
Total RNA was isolated using standard RNA extraction protocols (Trizol) and afterwards repurified by using the miRNeasy kit (Qiagen). RNA samples were quality-checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). RNA samples revealed RIN values between 6.6 and 9.4.
|
| Label |
Hy3
|
| Label protocol |
Sample labeling was performed according to the undisclosed miRXploreTM User manual. Control samples (miRXplore Universal Reference provided by Miltenyi ) were labeled with Hy3 and experimental samples were labeled with Hy5. The miRXplore Universal Reference represents a defined pool of synthetic microRNAs for comparison of multiple samples.
|
| |
|
| Channel 2 |
| Source name |
Non-Agressive carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Adrenal cortex
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using standard RNA extraction protocols (Trizol) and afterwards repurified by using the miRNeasy kit (Qiagen). RNA samples were quality-checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). RNA samples revealed RIN values between 6.6 and 9.4.
|
| Label |
Hy5
|
| Label protocol |
Sample labeling was performed according to the undisclosed miRXploreTM User manual. Control samples (miRXplore Universal Reference provided by Miltenyi ) were labeled with Hy3 and experimental samples were labeled with Hy5. The miRXplore Universal Reference represents a defined pool of synthetic microRNAs for comparison of multiple samples.
|
| |
|
| |
| Hybridization protocol |
The fluorescently labeled samples were hybridized overnight to miRXploreTM Microarrays using the a-HybTM Hybridization Station
|
| Scan protocol |
Fluorescence signals of the hybridized miRXploreTM Microarrays were detected using a laser scanner from Agilent (Agilent Technologies).
|
| Data processing |
Mean signal and mean local background intensities were obtained for each spot of the microarray images using the ImaGene software (Biodiscovery). Low-quality spots were flagged and excluded from data analysis. Unflagged spots were analysed with the PIQORTM Analyzer software. The PIQOR Analyzer allows automated data processing of the raw data text files derived from the ImaGene software. This includes background subtraction to obtain the net signal intensity, data normalization, and calculation of the Hy5/Hy3 ratios for the species of interest. As an additional quality filtering step, only spots/genes are taken into account for the calculation of the Hy5/Hy3 ratio that have a signal that is equal or higher than the 50% percentile of the background signal intensities.
For the identification of differentially regulated miRNA, the original dataset using the Universal Reference as hybridization control was used. All ratio data were transformed to logarithms to the base 2 (log2 ratio). For the discriminatory gene analysis (DGA) of the two different carcinoma subtypes, only miRNA were used which have valid expression values in at least four of the six samples provided for each subtype. 501 miRNA were retained after this filtering step. Regarding the DGA of adenoma and the combined carcinoma samples, at least six carcinoma samples out of twelve had to show valid expression values, while there had to be four out of six adenoma samples with valid expression values. 511 miRNA were retained after this filtering step. Heat maps showing filtered miRNA and their expression in the different cell samples relative to the Universal Reference were clustered using Euclidean distance and average linkage clustering. The log2 ratios were translated into a color code: red depicts a stronger expression of the miRNA in the sample relative to the control, green indicates a weaker expression, black stands for no differential regulation between sample and control and grey marks missing values.
|
| |
|
| Submission date |
Jan 04, 2013 |
| Last update date |
Jun 30, 2013 |
| Contact name |
Nadia Cherradi |
| E-mail(s) |
nadia.cherradi@cea.fr
|
| Phone |
+33 438 78 35 01
|
| Fax |
+33 438 78 50 58
|
| Organization name |
INSERM U1036
|
| Department |
iRTSV/CEA
|
| Lab |
Biology of Cancer and Infection
|
| Street address |
17 Rue des Martyrs
|
| City |
Grenoble Cedex 09 |
| ZIP/Postal code |
38054 |
| Country |
France |
| |
|
| Platform ID |
GPL16452 |
| Series (1) |
| GSE43279 |
MicroRNA profiling of benign and malignant adrenocortical tumors reveals potential biomarkers of recurrence |
|
