|
| Status |
Public on Apr 16, 2010 |
| Title |
TA 25 synapse |
| Sample type |
RNA |
| |
|
| Source name |
tibialis anterior muscle synapse
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: tibialis anterior muscle
cellular location: neuromuscular junction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and purified from each captured set by RNeasy micro kit (Qiagen, Valencia, CA) as described by manufacturer.
|
| Label |
Biotin
|
| Label protocol |
All total RNAs from EOM and TA synapse as well as EOM and TA fiber were used to generate double-stranded cDNA with the T7-oligo (dT) primer according to the GeneChip® Two cycle target labeling protocol (Affymetrix Inc., Santa Clara, CA, USA) as described by the manufacturer. At the end of the first cycle linear amplification, all amplified RNAs (i.e, aRNAs) were normalized to 600 ng (260/280 ratio ranging from 2.0-2.3) for the next amplification and labeling steps. Two rounds of in vitro transcription were performed as described by manufacturer. Labeled cRNA yield and purity were determined by measuring the absorbance at 260 and 280 nm using Nanodrop ND-1000 spectrophotometer.
|
| |
|
| Hybridization protocol |
Fifteen micrograms of labeled cRNA from TA synapse was fragmented, and 10μg was hybridized to Affymetrix® Rat 230 ver 2.0 GeneChip arrays for 18–24h (Affymetrix Inc.). Genechip was washed and stained with streptavidin–phycoerythrin.
|
| Scan protocol |
The chip was scanned at a 6μm resolution with Agilent model G2500A GeneArray scanner. A visual quality control measurement was performed to ensure proper hybridization after each chip was scanned.
|
| Description |
TA synapse was captured by LCM.
|
| Data processing |
Quality control parameters such as scaling factors used to normalize the chips, average background, and noise were also evaluated. In addition, raw intensities for each probe set were stored in electronic formats by the GeneChip Operating System version 1.1 (GCOS1.1, Affymetrix Inc.).
|
| |
|
| Submission date |
Apr 18, 2009 |
| Last update date |
Apr 23, 2009 |
| Contact name |
Murat T Budak |
| E-mail(s) |
[email protected]
|
| Phone |
215-8989726
|
| Organization name |
University of Pennsylvania
|
| Department |
|
| Lab |
Rubinstein and Khurana Labs
|
| Street address |
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL1355 |
| Series (1) |
| GSE15737 |
Fiber and NMJ / synaptic gene expression comparisons in rat extraocular muscle (EOM) and tibialis anterior (TA) muscle. |
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