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| Status |
Public on Aug 01, 2012 |
| Title |
MDSC, P1, WT mice, Isolation 1 |
| Sample type |
RNA |
| |
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| Source name |
Cultured cells, WT MDSC, Passage-1, Replicate1
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Hindlimb muscle as source of MDSC
gender: Male
age: 5 weeks
|
| Treatment protocol |
As the cells were plain growing cells and the aim of the experiment was to define the cell potency on the basis of gene expression signature, no treatment were given to MDSC or mESC.
|
| Growth protocol |
Cell isolation protocol. MDSC were isolated using a well established preplate technique based on cell adhesion properties from entire hindlimb muscles of WT and Myostatin null mice. The cells settle as different fractions called rapidly adhering populations(RAP) in the begining and slowly adhering cell poulations (SAP). RAPs are primarily fibroblasts follwed by myoblasts while SAPs are stem cells called MDSC. This protocol involves 0.2% collagenase 1 digestion of entire hind limb muscles of mice followed by their initial plating for 3 hours onto tissue culture dishes. After three hours, the settling down cell population is called Preplate1(PP1). The supernatant from PP1 was then transferred to a matrigel coated 10cm tissue culture dish and was allowed to settle for 24 hours. The adhering cell population is called Preplate2(PP2). Next day, after 24 hours,the supernatant from PP2 which is called PP3 was transferred to another matrigel coated 10 cm dish for adhesion for another 24 hours. Like this sequential transfer of supernatant was done every 24 hours till preplate 6( PP6). The adhering cell population from PP6 is called MDSC. MDSC at P1 was expanded and used for RNA isolation and current microarray gene expression analysis. In case of mouse ES cells, the W4 mES cell line from Taconic was received as a gift.These cells at P11 was revived on mitomycin C inactivated Mouse Embryonic Fibroblasts( MEF)/Feeders and then passaged till P15 on mitotically inactivated feeders.For RNA isolation of mESC, the W4 mESC growing on MEFs were trypsinized and plated onto gelatin coated dishes for 20 minutes so as to get rid of the MEFs.
MDSC isolated by preplate technique were grown on MDSC media consisting of DMEM, high glucose with 10% fetal bovine serum( Hyclone), 10% Horse serum(Gibco),!%Chicken Embryo Extract(US Biologicals),1% Penstep( Gibco) and 0.1mM cell culture grade β mercaptoethanol( Sigma) under 5% CO2 and humidified environment in a tissue culture incubator. W4 mESC were grown on mitotically inactivated MEFs in media containing DMEM High Glucose, 20% Fetal bovine serum(Hyclone),1% Non Essential Amino Acids(Gibco),1%Sodium pyruvate(Gibco),1%L-Glutamine(Gibco), 1% Penstep(Gibco) and Leukemia inhibitory factor , LIF(Millipore) at a concentration of 10^3 units per ml under 5% CO2 and humidified environment in a tissue culture incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using Qiagen RNeasy Mini kit as per manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of 70% confluent cells in culture
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| Submission date |
Jul 31, 2012 |
| Last update date |
Aug 01, 2012 |
| Contact name |
Bipasha Bose |
| E-mail(s) |
BBOSE@ntu.edu.sg, Bipasha.bose@gmail.com
|
| Organization name |
Nanyang Technological University
|
| Street address |
60, Nanyang Drive
|
| City |
Singapore |
| ZIP/Postal code |
637553 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE39765 |
Development of gene expression signature for defining the cell potency of muscle derived stem cells (MDSC) from mice of diffferent genotypes |
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