|
| Status |
Public on Jul 23, 2015 |
| Title |
CD11b+ cells MCA203_2-mirna |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MCA203_2-mirna
|
| Organism |
Mus musculus |
| Characteristics |
disease state: Tumour-bearing
source tissue: Tumour infiltrate
strain: C57BL/6
cell type: CD11b+
|
| Treatment protocol |
CD11b+ cells were isolated by immuno-sorting using CD11b+ isolation kit (Miltenyi Biotech) and LS columns.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from CD11b+ sorted cells by TRIzol (Invitrogen) according to the manufacturer’s instructions. The quality and quantity of RNA samples were determined by Agilent RNA 6000 Nano Chip (Agilent Technologies). Before hybridization, total RNA was size-fractioned to enrich in small RNA (minus than 70 nt in length) with the FlashPAGE purification kit (Ambion), according to the manufacturer’s instructions.
|
| Label |
cy5
|
| Label protocol |
Cy5 Sample Labeling Protocol. Small RNA was enriched from 10ug total RNA by flashPAGE (Pre-cast Gel,Type A) (Ambion, Austin, TX, USA) and purified using flashPAGE reaction clean-up kit (Ambion, Austin, TX, USA) according to manufacture’s instruction. The same procedures were applied to obtain small RNA from the Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines that was used as the reference for the miRNA expression array assay. Fragmented small RNA were 3’-end tailed with amine-modified nucleotides and chemically coupled to CyDye fluors (Amersham Biosciences, piscatway, NJ, USA), which sample with Cy5 and the reference with Cy3 respectively, using the mirVana miRNA Labeling Kit (Ambion) following the recommended protocol by the manufactures’ instruction. After labeling, the samples and the reference were co-hybridized to the miRNA array at room temperature over night. Both the processed cDNA and the miRNA array slides were scanned by GenePix scanner Pro 4.0 (Axon, Sunnyvale, CA, USA).
|
| |
|
| Channel 2 |
| Source name |
Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol
|
| Label |
Cy3
|
| Label protocol |
Small RNA was enriched from 10ug total RNA by flashPAGE (Pre-cast Gel,Type A) (Ambion, Austin, TX, USA) and purified using flashPAGE reaction clean-up kit (Ambion, Austin, TX, USA) according to manufacture’s instruction. The same procedures were applied to obtain small RNA from the Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines that was used as the reference for the miRNA expression array assay.
|
| |
|
| |
| Hybridization protocol |
After labeling, the samples and the reference were co-hybridized to the miRNA array at room temperature over night. Both the processed cDNA and the miRNA array slides were scanned by GenePix scanner Pro 4.0 (Axon, Sunnyvale, CA, USA).
|
| Scan protocol |
Creator:GenePix Pro 4.0.1.17 Scanner:GenePix 4000B [84945]
|
| Description |
CD11b+ cells from tumor infiltrate of MCA203 tumor-bearing mice
|
| Data processing |
BRBArrayTools Calculation Method: Fluorescence intensities generated by Cy5 and Cy3 probes hybridized onto the microarray slides were scanned were scanned on a GenePix 4000 (Axon Instruments, Union City, CA) at variable PMT voltage to obtain maximal signal intensities with < 1% probe saturation. Following filter criteria was applied to the data set: 1. spots were excluded if intensity < 100, and spot size less than 10um 2. genes were filtered out if >80% of experiment with missing data values. 3. log2 transformed data were normalized using lowess smoother, and intensity ratios were truncated if greater than 64.
|
| |
|
| Submission date |
Aug 01, 2012 |
| Last update date |
Jul 23, 2015 |
| Contact name |
Stefania Bortoluzzi |
| E-mail(s) |
stefania.bortoluzzi@unipd.it
|
| Phone |
++30 049 827 6502
|
| Organization name |
University of Padova
|
| Department |
Department of Molecular Medicine
|
| Lab |
Computational Genomics
|
| Street address |
via G. Colombo 3
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6773 |
| Series (1) |
| GSE39807 |
Gene and microRNA expression data from tumor induced CD11b+ MDSC |
|
