|
| Status |
Public on Nov 28, 2007 |
| Title |
SHR rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Primary branch of saphenous artery pooled from 2-3 16 week old male spontaneously hypertensive (SHR) rats
|
| Organism |
Rattus norvegicus |
| Characteristics |
16 week old male spontaneously hypertensive (SHR) rats
|
| Treatment protocol |
no treatment
|
| Growth protocol |
All RNA samples were obtained from male 16-week old Wistar-Kyoto rats or male 16-week old SHR rats
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Rats were anesthetized with ether and killed by decapitation. A primary branch of the saphenous artery was removed, immersed in RNAlater (Ambion) and extraneous material removed in cold phosphate-buffered saline (PBS). Samples (n=3) were taken from 2 or 3 animals from each experimental group. Arteries were snap frozen in liquid nitrogen and processed using the RNeasy Micro Kit (Qiagen), including a 20 min DNAse treatment. RNA quality was monitored on an Agilent 2100 Bioanalyzer using the RNA 6000 Pico LabChip kit (Agilent). Concentrations of RNA were measured by UV spectrophometry.
|
| Label |
biotin
|
| Label protocol |
Two cycle preparation of cRNA and hybridization: Sample preparation followed the Affymetrix GeneChip Eukaryotic Small Sample Target Labeling Assay Version II [42]. Double-stranded cDNA was synthesized using oligo(dT) 24-anchored T7 primer, Superscript III (Invitrogen) and 100ng total RNA for the first strand followed by DNA polymerase I and T4 DNA polymerase for the second strand. The double-stranded cDNA was precipitated with ethanol, air-dried and used as template for the first round of in vitro transcription of cRNA with MEGAscript enzyme mix (Ambion) at 37ºC for 7 h. The cRNA was purified (RNeasy, Qiagen) and used as template for the second round of double-stranded cDNA synthesis with random primers (Invitrogen). The double-stranded cDNA product of second round synthesis was purified by ethanol precipitation and used as template for the second round of in vitro transcription and labeling of cRNA, using the ENZO BioArray HighYield RNA Transcript Labeling kit (Affymetrix) according to the protocol. All components were mixed, incubated at 37ºC for 7 h, and the biotin-labeled cRNA was purified (RNeasy, Qiagen) and fragmented prior to hybridisation to Affymetrix GeneChip RAE230A arrays. Hybridization, washing and staining followed the Affymetrix GeneChip Expression Analysis protocols. Data were collected and analyzed using the program MAS5 (Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C to each array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400. Affymetrix GeneChip Expression Analysis protocols were used throughout.
|
| Scan protocol |
GeneChips were scanned using an Affymetrix 2500A GeneArray Scanner.
|
| Description |
Gene expression data from a primary branch of the saphenous artery of 16 week old spontaneously hypertensive (SHR) rats
|
| Data processing |
Data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as the normalization method. The trimmed mean target intensity of each array was set to 150.
|
| |
|
| Submission date |
Jun 07, 2007 |
| Last update date |
Nov 28, 2007 |
| Contact name |
Stephen John Ohms |
| E-mail(s) |
[email protected]
|
| Phone |
61-2-6125-2522
|
| Organization name |
Australian National University
|
| Department |
John Curtin School of Medical Research
|
| Lab |
ACRF Biomolecular Resource Facility
|
| Street address |
Garran Road
|
| City |
Canberra |
| State/province |
Australian Capital Territory |
| ZIP/Postal code |
GPO Box 334, Canberra City |
| Country |
Australia |
| |
|
| Platform ID |
GPL341 |
| Series (1) |
| GSE8051 |
Gene expression in a resistance artery in 2 models of hypertension in the rat |
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