|
| Status |
Public on Feb 10, 2012 |
| Title |
13.5dpc CA vs 12.5dpc MC (Dye Combination 2) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cartilage anlagen, 13.5dpc hind limb
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Cartilage anlagen hind limb
age: 13.5dpc
strain: Swiss white
|
| Treatment protocol |
Dissected limb buds were embedded in Tissue-Tek OCT (Sakura Fine Technical), snap-frozen in isopentane, and stored at -80° C. 6um cryosections were prepared using a cryostat (Leica CM1850), mounted on RNAse-free SuperFrost Plus slides (Biolab Scientific), and processed immediately by being fixed in 70% ethanol, rinsed in RNAse-free water, and dehydrated in 70%, 95%, and 100% ethanol for 30 seconds each. They were then stored in 100% ethanol at -80° C. Immediately prior to microdissection, sections were air-dried and the tissue of interest was microdissected using an ophthalmic scalpel (Feather) fixed to the scanning xy-object guide. Light microscopy was performed using an Eclipse 80i light microscope (Nikon).
|
| Growth protocol |
Pregnant Swiss white mice were sacrificed in accordance with institutional ethical guidelines at the appropriate stage of gestation. Embryos were then harvested and culled, and hind limb buds were dissected.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Microdissected tissues were collected in TRIzol reagent (Invitrogen) for RNA extraction and purification, which was performed following the manufacturer’s specifications. All total RNA samples were subjected to two rounds of linear amplification using the MessageAmp II aRNA Amplification Kit (Ambion), following the manufacturer’s protocol. 100-150ng of first-round amplified aRNA was used as template for each second round amplification. Following total RNA extraction and amplification, the yield, purity, and integrity of all RNA samples were validated by capillary electrophoresis with a Bioanalyzer 2100 (Agilent Technologies), using a Series II RNA 6000 Pico Kit (Agilent Technologies), according to the manufacturer’s specifications.
|
| Label |
Cy3
|
| Label protocol |
Amplified RNA samples (2ug) were labelled with Cy3 or Cy5 (Amersham), according to the manufacturer’s specifications. Fluorophore incorporation and yield were determined by spectrophotometry using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| Channel 2 |
| Source name |
Mesenchymal condensation, 12.5dpc hind limb
|
| Organism |
Mus musculus |
| Characteristics |
strain: Swiss white
tissue: Mesenchymal condensation hind limb
age: 12.5dpc
|
| Treatment protocol |
Dissected limb buds were embedded in Tissue-Tek OCT (Sakura Fine Technical), snap-frozen in isopentane, and stored at -80° C. 6um cryosections were prepared using a cryostat (Leica CM1850), mounted on RNAse-free SuperFrost Plus slides (Biolab Scientific), and processed immediately by being fixed in 70% ethanol, rinsed in RNAse-free water, and dehydrated in 70%, 95%, and 100% ethanol for 30 seconds each. They were then stored in 100% ethanol at -80° C. Immediately prior to microdissection, sections were air-dried and the tissue of interest was microdissected using an ophthalmic scalpel (Feather) fixed to the scanning xy-object guide. Light microscopy was performed using an Eclipse 80i light microscope (Nikon).
|
| Growth protocol |
Pregnant Swiss white mice were sacrificed in accordance with institutional ethical guidelines at the appropriate stage of gestation. Embryos were then harvested and culled, and hind limb buds were dissected.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Microdissected tissues were collected in TRIzol reagent (Invitrogen) for RNA extraction and purification, which was performed following the manufacturer’s specifications. All total RNA samples were subjected to two rounds of linear amplification using the MessageAmp II aRNA Amplification Kit (Ambion), following the manufacturer’s protocol. 100-150ng of first-round amplified aRNA was used as template for each second round amplification. Following total RNA extraction and amplification, the yield, purity, and integrity of all RNA samples were validated by capillary electrophoresis with a Bioanalyzer 2100 (Agilent Technologies), using a Series II RNA 6000 Pico Kit (Agilent Technologies), according to the manufacturer’s specifications.
|
| Label |
Cy5
|
| Label protocol |
Amplified RNA samples (2ug) were labelled with Cy3 or Cy5 (Amersham), according to the manufacturer’s specifications. Fluorophore incorporation and yield were determined by spectrophotometry using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| |
| Hybridization protocol |
All aRNA samples were hybridized to 44K mouse whole genome microarrays (Agilent Technologies), in two color hybridizations including dye swaps, and in strict accordance with the manufacturer’s specifications.
|
| Scan protocol |
All microarrays were scanned at 10um resolution on an Axon 4000B scanner
|
| Data processing |
The microarray features were acquired using GenePix Pro 4.1 software, and the raw data was processed using a print tip loess method of normalization with limmaGUI, which is an implemented package of R used to fit linear models to microarray data.
|
| |
|
| Submission date |
Feb 09, 2012 |
| Last update date |
Feb 10, 2012 |
| Contact name |
Trevor L Cameron |
| E-mail(s) |
trevor.cameron@mcri.edu.au
|
| Organization name |
Murdoch Childrens Research Institute
|
| Street address |
Flemington Road
|
| City |
Parkville |
| ZIP/Postal code |
3052 |
| Country |
Australia |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE35669 |
Global comparative transcriptome analysis of cartilage formation in vivo |
|
