|
| Status |
Public on Mar 15, 2019 |
| Title |
4c_NKG2DLneg |
| Sample type |
RNA |
| |
|
| Source name |
CD34 non-expressing AML, sorted fraction: NKG2DLneg
|
| Organism |
Homo sapiens |
| Characteristics |
origin: AML, peripheral blood, CD33+
sorted fraction: NKG2DLneg
|
| Treatment protocol |
For simultaneous staining of all NKG2DL, a recombinant NKG2D-Fc chimera (fusion protein) and the corresponding isotype control were purchased from R&D (Minneapolis, MN, USA) and biotinylated with the One-step biotinylation kit (MACS Miltenyi) according to manufactures instructions. Prior to staining, cells were blocked with human IgG (Sigma-Aldrich) and washed. Then biotinylated fusion protein or control (10µg/ml each) were added followed by two washing steps. Afterwards streptavidin-PE (LifeTechnologies, Carlsbad, CA, USA) and selection antibodies were further added prior to sorting into NKG2DLpos and NKG2DLneg fractions
|
| Growth protocol |
Standard protocol for primary human tissue
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and RNA concentration and purity determined on the NanoDrop ND-1000 spectral photometer (peqlab). The 2100 Bioanalyzer (Agilent Technologies) and the RNA 6000 Nano as well as the 6000 Pico LabChip Kit (Agilent Technologies) were used to analyze RNA integrity.
|
| Label |
Cy3
|
| Label protocol |
100 ng total RNA per sample were introduced into a reverse transcription with subsequent in vitro transcription (RT-IVT) reaction. Prior to RT-IVT, the total RNA samples were spiked with in vitro synthesized polyadenylated transcripts (One-Color RNA Spike-In Mix, Agilent Technologies) which serve as an internal labeling control for linearity, sensitivity and accuracy. The spiked total RNA was reverse transcribed into cDNA and then converted into Cyanine-3 labeled cRNA (Low Input Quick-Amp Labeling Kit One-Color, Agilent Technologies). All steps were carried out according to manufacturer’s instructions. cRNA concentration (ng/μl), RNA absorbance ratio (A260nm/A280nm) and Cyanine-3 dye concentration (pmol/μl) were recorded for all cRNA samples using NanoDrop ND-1000 UV- VIS spectrophotometer. Yield and specific activity of each reaction were determined. The RNA 6000 Nano LabChip Kit (Agilent Technologies) was used on the 2100 Bioanalyzer to analyze the quality of labeled non-fragmented cRNA.
|
| |
|
| Hybridization protocol |
Following cRNA clean-up and quantification 600 ng of each Cyanine-3-labeled cRNA sample was fragmented and prepared for one-color-based hybridization (Gene Expression Hybridization Kit, Agilent Technologies). Labeled cRNA samples were hybridized at 65°C for 17 hrs on separate Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays (AMADID 039494). Afterwards, microarrays were washed with increasing stringency using Gene Expression W ash Buffers (Agilent Technologies) followed by drying with acetonitrile (SIGMA).
|
| Scan protocol |
Fluorescent signal intensities were detected with Scan Control A.8.4.1 software (Agilent Technologies) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.7.3.1 software (Agilent Technologies).
|
| Data processing |
The software tools Feature Extraction 10.7.3.1, GeneSpring GX 12.6 (both Agilent Technologies), Excel 2010 (Microsoft) and the IMGM internal tool marfin v1.9 were used for quality control, statistical data analysis, filtering of differentially expressed transcript, RNA annotation and visualization.
|
| |
|
| Submission date |
Feb 26, 2019 |
| Last update date |
Mar 16, 2019 |
| Contact name |
Claudia Lengerke |
| E-mail(s) |
[email protected]
|
| Organization name |
University Hospital Basel and University of Basel
|
| Department |
Department of Biomedicine
|
| Street address |
Hebelstr. 20
|
| City |
Basel |
| ZIP/Postal code |
4055 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE127200 |
Gene expression data from sorted primary human acute myeloid leukemia (AML) samples |
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