|
Status |
Public on Apr 09, 2010 |
Title |
Intervertebral disc-mutant-2 |
Sample type |
RNA |
|
|
Source name |
IVD E13.5 days, mutant
|
Organism |
Mus musculus |
Characteristics |
tissue: Intervertebral disc (IVD) genotype: Cre+;Tgfbr2lox/lox
|
Treatment protocol |
Comparison of control and Col2aCre;Tgfbr2 deleted mice.
|
Growth protocol |
Mouse embryos, embryonic day E13.5 day.
|
Extracted molecule |
total RNA |
Extraction protocol |
E13.5 day control and mutant mouse embryos were rinsed in DEPC treated PBS, embedded into OCT and frozen for sectioning. Using a cryostat, 8-12um sagittal cut frozen sections were collected and placed on PALM PEN-Membrane Slides (P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany). The frozen sections were then quickly dehydrated (70 to 100% EtOH) and stored in Xylene prior to LCM. Laser Capture Microdissection (LCM) was carried out by using a Zeiss/ PALM Microbeam Instrument (Microdissection System; Carl Zeiss Microimaging GmbH, Munchen, Germany). The presumptive IVD from the lumbar region were collected into RNase/DNase free special PALM AdhesiveCaps (P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany). After IVDs were collected, the adjacent presumptive vertebrae were collected into a separate adhesive cap. Collected sample tubes are stored at -80C until RNA was isolated. RNA was isolated using Ambion RNAqueous – Micro Kit (Austin, TX). The optional DNase treatment step was included.
|
Label |
biotin
|
Label protocol |
Detailed genechip analysis procedures are presented in the Manufacturer’s GeneChip Expression Technical Manual (Affymetrix). Briefly, 50ng of total RNA from each sample was used in a two cycle cDNA amplification protocol using T7-linked oligo dT primers as per the manufacturer’s instructions. After the first round of cDNA synthesis an in vitro transcription step was utilized to amplify the RNA following which a second round of cDNA synthesis was performed. Subsequently, cRNA was generated and biotin was incorporated into the cRNA strand by standard methods (Affymetrix) followed by cRNA fragmentation, and preparation of hybridization cocktail.
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Hybridization protocol |
Detailed genechip analysis procedures are presented in the Manufacturer’s GeneChip Expression Technical Manual (Affymetrix). The arrays were hybridized overnight at 45oC, and then washed, stained, and scanned the next day.
|
Scan protocol |
Detailed genechip analysis procedures are presented in the Manufacturer’s GeneChip Expression Technical Manual (Affymetrix). Gene expression levels were extracted using AGCC (Affymetrix GeneChip Command Console).
|
Description |
Intervertebral disc-mutant-2
|
Data processing |
The data was analyzed with Genesprings using the default settings. The summarized probeset was generated using RMA summarization algorithm. Quantile normalization was used. Baseline transformation to median was used.
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|
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Submission date |
Oct 20, 2009 |
Last update date |
Aug 28, 2018 |
Contact name |
Rosa Serra |
E-mail(s) |
rserra@uab.edu
|
Phone |
205-934-0842
|
Organization name |
University of Alabama at Birmingham
|
Department |
Cell Biology
|
Lab |
Serra
|
Street address |
1918 University Blvd. 660 MCLM
|
City |
Birmingham |
State/province |
AL |
ZIP/Postal code |
35294-0005 |
Country |
USA |
|
|
Platform ID |
GPL1261 |
Series (2) |
GSE18647 |
Gene expression in embryonic intervertebral disc and vertebrae. |
GSE18649 |
Molecular Profiling of the Developing Axial Skeleton: A role for Tgfbr2 in the Development of the Intervertebral Disc. |
|
Relations |
Reanalyzed by |
GSE119085 |