|
| Status |
Public on Oct 21, 2008 |
| Title |
SMC 10 uM homocysteine rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Aortic smooth muscle cells
|
| Organism |
Homo sapiens |
| Characteristics |
CC-2571 cell line Cambrex-Lonza
|
| Biomaterial provider |
Cambrex - Lonza
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cells using the RNeasy Mini kit (Qiagen) and analysed on the HP 2100 Bioanalyzer (Agilent Technologies) for integrity
|
| Label |
biotinylated UTP
|
| Label protocol |
Biotin-labeled cRNA was prepared by linear amplification of the Poly(A)+ RNA population within the total RNA sample. Briefly, 0.44 µg of total RNA was reverse transcribed after priming with a DNA oligonucleotide containing T7 RNA polymerase promoter 5' to a d(T)24 sequence. After second-strand cDNA synthesis and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase in the presence of biotinylated UTP.
|
| |
|
| Hybridization protocol |
10 µg of purified cRNA was fragmented to uniform size and applied to CodeLink Bioarrays (GE Healthcare) in hybridization buffer. Arrays were hybridized at 37ºC for 18 hrs in a temperature-controlled shaking incubator. Arrays were washed in 0.75x TNT at 46º C for 1 hr and stained with Cy5-Streptavidin dye conjugate for 30 min.
|
| Scan protocol |
Arrays were scanned with a GenePix™ 4000B scanner.
|
| Description |
GE Healthcare Amersham CodeLink™ iExpress Assay Reagent Kit
Product booklet Codes: 67601000
4. Description
4.1 CodeLink system summary:
CodeLink™ Expression Bioarray System comprises a set of bioarray
products and tools for gene expression profi ling experiments that
allows monitoring of the mRNA levels of multiple genes
simultaneously. The system includes:
• sets of carefully designed and validated bioarrays with
integrated hybridization chambers that cover a wide range of
discovery genes for several organisms
• cRNA target preparation and hybridization reagents
• optimized protocols for cRNA target preparation and bioarray
processing
• hybridization and post-hybridization parallel processing tools
and fi xtures
• bioarray quantitation analysis software
4.2. Protocol overview
4.2.1. cRNA target preparation
The biotin-labelled cRNA target is prepared by a linear amplifi cation
method. The poly(A)+ RNA (mRNA) subpopulation within the total RNA
population is primed for reverse transcription by a DNA oligonucleotide
containing the T7 RNA polymerase promoter 5’ to a d(T)24 sequence. After second-strand cDNA synthesis, the cDNA serves as the
template for an in vitro transcription (IVT) reaction to produce the
target cRNA. The IVT is performed in the presence of biotinylated
nucleotides to label the target cRNA. This method produces
approximately 1 000- to 5 000-fold linear amplifi cation of the input
mRNA.
A set of bacterial mRNA controls is included in each CodeLink iExpress
Assay Reagent Kit to serve as an overall platform performance
control group, and can also be used to estimate the sensitivity of RNA
detection.
13
An independent total RNA control (HeLa) and an exogenous cDNA
IVT control (pTRI-Xef 1 plasmid) are also included in each CodeLink
iExpress Assay Reagent Kit. The HeLa RNA can be used to monitor
overall kit performance; the pTRI-Xef 1 plasmid can be used to
evaluate IVT performance.
4.2.2. Target hybridization and bioarray processing
Hybridization is performed overnight in a temperature-controlled
shaking incubator. Optimized hybridization buffer components
are included in CodeLink iExpress Assay Reagent Kit for use at this
step. The instructions for performing the hybridization and posthybridization
processing steps and bioarray processing are provided
in the CodeLink Gene Expression System: Single-Assay Bioarray
Hybridization and Detection protocol or the CodeLink Gene
Expression System: 16-Assay Bioarray Hybridization and Detection
protocol, depending on the bioarray format to be used.
Post-hybridization processing includes a stringent wash to remove
unbound and non-specifi cally hybridized target molecules, a
secondary-labeling step using a Cy™5-Streptavidin conjugate, and
several washing steps to remove unbound conjugate. Following a
final rinse, the bioarrays are dried by centrifugation and scanned.
Analysis of the bioarrays was done usingwith the CodeLink Expression Analysis software.
|
| Data processing |
Array data was processed with CodeLink Expression Analysis software (GE Healthcare) and data was analyzed with GeneSpring software (Silicon Genetics). To compare individual expression values across arrays, raw intensity data (generated from CodeLink Expression 2 software) from each gene was normalized to the median intensity of the array. Only genes which had values greater than the background intensity in at least one condition were used for further analysis.
|
| |
|
| Submission date |
Oct 31, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Jonathan Golledge |
| E-mail(s) |
jonathan.golledge@jcu.edu.au
|
| Phone |
+67 7 47814730
|
| Fax |
+61 7 4781 6986
|
| URL |
http://www.jcu.edu.au/school/medicine/VBU.htm
|
| Organization name |
James Cook University
|
| Department |
School of Medicine
|
| Lab |
Vascular Biology Unit
|
| Street address |
School of Medicine
|
| City |
Townsville |
| State/province |
Queensland |
| ZIP/Postal code |
4811 |
| Country |
Australia |
| |
|
| Platform ID |
GPL2895 |
| Series (1) |
| GSE9490 |
The effect of homocysteine on human aortic smooth muscle cells |
|
