|
| Status |
Public on Jun 24, 2011 |
| Title |
RV sham-Control vs. RV following banding the pulmonary artery (PAB-RV) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Right venticluar heart tissue (male, 15-20 weeks; 3 to 4 kg)
|
| Organism |
Oryctolagus cuniculus |
| Characteristics |
sample type: control
gender: male
age: 15-20 weeks
weight: 3 to 4 kg
tissue: Right ventricular heart
treatment: RV sham-Control
|
| Treatment protocol |
Newborn New Zealand White rabbits were sedated and anesthetized and a mini thoracotomy was performed. LV-POH was achieved by banding the descending thoracic aorta (n=6), RV-POH was achieved by banding the pulmonary artery (n=6). Sham–Control animals (SC; n=6 each) were sham-manipulated. Following 4 (LV-POH) and 6 weeks (RV-POH) recovery the hearts were removed and matched sample RNA and proteins were isolated for microarray and proteomic analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cardiac ventricular RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
4uL first strand buffer; 2uL 0.1M DTT; 2uL 10X low dTTP–dNTP (5 mM dATP, dCTP, dGTP, and 2 mM dTTP); 2uL CyanineX-dUTP (1 mM ; Cyanine3; Cy3 for control; green and Cyanine5; Cy5 for experimental; Amersham, Pharmacia Biotech); 1uL RNasin and 1uL of Superscript II (GIBCO) and incubated at 42 oC for 30 min. then and another 1uL Superscript II added and the reaction allowed to continue a further 40 min. at 42 oC. The reaction was stopped with the addition of 2.5 uL 500 mM EDTA and heating to 65 oC for 1 min. The RNA template was hydrolyzed with the addition of 5 uL 1M NaOH and incubation at 65 oC for 10 min. and then neutralized by adding 12.5 uL 1M Tris pH 7.5 immediately to neutralize the pH. The labeled cDNA probes were purified by gel exclusion chromatography (ProbeQuant G-50, Amersham) and the volume reduced to 5-10 uL using a Speedvac.
|
| |
|
| Channel 2 |
| Source name |
Right ventricular heart tissue (male,15-20 weeks; 3 to 4 kg)
|
| Organism |
Oryctolagus cuniculus |
| Characteristics |
sample type: test
gender: male
age: 15-20 weeks
weight: 3 to 4 kg
tissue: Right ventricular heart
treatment: RV following banding the pulmonary artery (PAB-RV)
|
| Treatment protocol |
Newborn New Zealand White rabbits were sedated and anesthetized and a mini thoracotomy was performed. LV-POH was achieved by banding the descending thoracic aorta (n=6), RV-POH was achieved by banding the pulmonary artery (n=6). Sham–Control animals (SC; n=6 each) were sham-manipulated. Following 4 (LV-POH) and 6 weeks (RV-POH) recovery the hearts were removed and matched sample RNA and proteins were isolated for microarray and proteomic analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cardiac ventricular RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
4uL first strand buffer; 2uL 0.1M DTT; 2uL 10X low dTTP–dNTP (5 mM dATP, dCTP, dGTP, and 2 mM dTTP); 2uL CyanineX-dUTP (1 mM ; Cyanine3; Cy3 for control; green and Cyanine5; Cy5 for experimental; Amersham, Pharmacia Biotech); 1uL RNasin and 1uL of Superscript II (GIBCO) and incubated at 42 oC for 30 min. then and another 1uL Superscript II added and the reaction allowed to continue a further 40 min. at 42 oC. The reaction was stopped with the addition of 2.5 uL 500 mM EDTA and heating to 65 oC for 1 min. The RNA template was hydrolyzed with the addition of 5 uL 1M NaOH and incubation at 65 oC for 10 min. and then neutralized by adding 12.5 uL 1M Tris pH 7.5 immediately to neutralize the pH. The labeled cDNA probes were purified by gel exclusion chromatography (ProbeQuant G-50, Amersham) and the volume reduced to 5-10 uL using a Speedvac.
|
| |
|
| |
| Hybridization protocol |
Microarray slides were pre-hybridized in a solution containing 3.5 x SSC, 0.1% SDS and 1% BSA at 50C for at least 60 min. and then rinsed 2x in ddH2O; once in isopropanol and then spun dry at 1,000 rpm for 1 min and then immediately used for hybridization. Control heart samples were labeled with Cy3-dUTP and experimental hearts samples (AOB-LV and PAB-RV) were labeled with Cy5-dUTP and then combined with 30 uL hybridization solution (stock solution containing 100 uL DIG EasyHyb hybridization solution (Roche), 5 uL yeast tRNA (10 mg/mL), 5 uL human COT1 DNA, 5 uL Poly-dA, and 5 uL salmon sperm DNA (10 mg/mL). Dye swaps also performed. The probe solution was hybridized to the arrayed slide at 37 C overnight. The following day, slides were washed first with 1x SSC to remove the cover slip (Hybri-Slips, Sigma) , then 3 successive washes of 1x SSC / 0.1% SDS for 15 minutes each at 50 oC, followed by a rinse with 1x SSC. The slides were dried in individual 50 mL conical tubes and centrifuged at 500 rpm to remove excess fluid.
|
| Scan protocol |
Scanning of the slides was performed using the GMS 418 scanner (Affymetrix) at 532 nm (Cy3) and 635 nm (Cy5).
|
| Description |
Rabbit Heart right ventricle (RV) sham-Control vs. Rabbit Heart right ventricle (RV) following banding the pulmonary artery (PAB-RV)
|
| Data processing |
Local background was subtracted from the fluorescent value Cluster Analysis. All treatments were compared to Control to account for constitutive gene expression. Raw scanned images of Cy3 and Cy5 fluorescence were processed using Axon 4000A microarray scanner with GenePix analysis software using Rosetta Resolver (Molecular Devices Corp, Sunnyvale CA.). Dye swap was treated as technical replicates to account for dye effect error. Fold change criteria was used together with p-values to identify gene expression changes. p values were calculated based on dye swap results. Computational biology was performed using Spotfire Decision Site 8.1 (Spotfire, Somerville, MA). Functional annotation cluster analysis was performed using DAVID 2007 functional annotation bioiformatics microarray analysis (http://david.abcc.ncifcrf.gov/).
Lowess normalization.
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| |
|
| Submission date |
Jun 24, 2011 |
| Last update date |
Jun 24, 2011 |
| Contact name |
James D McCully |
| E-mail(s) |
James_mccully@hms.harvard.edu
|
| Phone |
617-667-0725
|
| Fax |
617-975-5245
|
| Organization name |
BIDMC/Harvard Medical School
|
| Department |
Cardiothoracic Surgery
|
| Street address |
77 Avenue Louis Pasteur
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL13758 |
| Series (1) |
| GSE30194 |
Rabbit heart gene expression in Left ventricle (LV) following banding of the descending thoracic aorta (AOB-LV) vs Sham-control LV and in Right ventricle (RV) following banding the pulmonary artery (PAB-RV) vs. Sham-control RV |
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