|
| Status |
Public on Sep 05, 2013 |
| Title |
Control B220_vs_K1 B220 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control B220
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype: Blimp-1 flox and CD11c-CRE-
gender: female
age: 6-10 weeks
tissue: spleen
cell type: B220+ B cells
|
| Treatment protocol |
Spleens were dissociated by 40 U/ml of collagenase D. Total splenocytes were stained with antibodies (anti-CD11c, anti-MHC II, anti-CD4, anti-TCRb, and anti-B220) on ice for 15 min. Each population of cells was purified by a cell sorter, spun down and snap frozen by liquid nitrogen until shipping to the company.
|
| Growth protocol |
No additional growth was done.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol.
|
| Label |
Hy3
|
| Label protocol |
1.5 µg (for 0007090032 - 0007090035) and 2 µg (for 0007090036 and 0007090037) of the respective total RNA were each mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled using a commercial kit (miRCuryTM LNA microRNA Array Power labeling kit, Exiqon) following the instructions of the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
KO B220
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype: Blimp-1 flox and CD11c-CRE+
gender: female
age: 6-10 weeks
tissue: spleen
cell type: B220+ B cells
|
| Treatment protocol |
Spleens were dissociated by 40 U/ml of collagenase D. Total splenocytes were stained with antibodies (anti-CD11c, anti-MHC II, anti-CD4, anti-TCRb, and anti-B220) on ice for 15 min. Each population of cells was purified by a cell sorter, spun down and snap frozen by liquid nitrogen until shipping to the company.
|
| Growth protocol |
No additional growth was done.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol.
|
| Label |
Hy5
|
| Label protocol |
1.5 µg (for 0007090032 - 0007090035) and 2 µg (for 0007090036 and 0007090037) of the respective total RNA were each mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled using a commercial kit (miRCuryTM LNA microRNA Array Power labeling kit, Exiqon) following the instructions of the manufacturer.
|
| |
|
| |
| Hybridization protocol |
The fluorescently labeled samples were hybridized overnight to miRXplore TM Microarrays CTID 878 in a dual colour approach using the a-HybTM Hybridization Station (Miltenyi Biotec).
|
| Scan protocol |
Image capture and signal quantification of hybridized miRXplore ™ microarrays was done with the Agilent's Microarray Scanner System (Agilent Technologies, Palo Alto, USA) and ImaGene software Version 8.0.1 (BioDiscovery, Los Angeles, CA, USA).
|
| Description |
0007090032
|
| Data processing |
Signal quantification of the scanned miRXplore TM microarrays was done using ImaGene software Version 8.0.1 (BioDiscovery, Los Angeles, CA, USA). Local background was subtracted from the signal to obtain the net signal intensity and the ratio of Hy5/Hy3. Subsequently, the mean of the ratios of 4 corresponding spots representing the same miRNA was computed. The mean ratios were normalised using LOWESS.
A ratio was not calculated if the average signal intensities for both channels was below the adjusted threshold value (calculated based on the 50% percentile of the background signal intensities and adjusted by the mean of the calibration oligos):
avg(Cy5-bkg) < adjust_Cy5_default AND avg(Cy3-bkg) < adjust_Cy3_default
|
| |
|
| Submission date |
Sep 04, 2012 |
| Last update date |
Sep 05, 2013 |
| Contact name |
Silvia Rueberg |
| E-mail(s) |
silvia@miltenyibiotec.de
|
| Phone |
+49 (0)2204 8306 4219
|
| Organization name |
Miltenyi Biotec GmbH
|
| Department |
Genomic Services Department
|
| Street address |
Friedrich-Ebert-Str. 68
|
| City |
Bergisch Gladbach |
| ZIP/Postal code |
51429 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15979 |
| Series (1) |
| GSE40587 |
miRNA expression in Blimp-1-deficient dendritic cells |
|
