Please see our publication Wilson et al PLoS One 2008, PMID: 18648539
Growth protocol
N/A
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated separately from five heart samples for each condition using TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer's instructions. Total RNA was purified using RNeasy columns (Qiagen, Chatsworth, CA). RNA concentration was measured by spectrophotometry, and RNA integrity assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) with 6000 Nano Chips according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to 18S and 28S ribosomal RNA subunits and had a RNA Integrity Number (RIN) greater than six.
Label
Cy5
Label protocol
Using Low RNA Input Fluorescent Linear Amplification Kits (Agilent Technologies, Santa Clara, CA, USA), cDNA was reverse transcribed from each RNA sample, and cRNA was then transcribed and fluorescently labeled from each cDNA sample. The mouse heart tissue cRNA samples were labeled with Cy5 and the Universal Mouse reference cRNA was labeled with Cy3. The resulting cRNA was purified using an RNeasy kit (Qiagen, Valencia, CA, USA) followed by quantification of the cRNA by spectroscopy using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). 825 ng Cy3- and Cy5- labeled and amplified cRNA was mixed and fragmented according to the Agilent technology protocol.
Please see our publication Wilson et al PLoS One 2008, PMID: 18648539
Growth protocol
N/A
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated separately from five heart samples for each condition using TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer's instructions. Total RNA was purified using RNeasy columns (Qiagen, Chatsworth, CA). RNA concentration was measured by spectrophotometry, and RNA integrity assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) with 6000 Nano Chips according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to 18S and 28S ribosomal RNA subunits and had a RNA Integrity Number (RIN) greater than six.
Label
Cy3
Label protocol
Using Low RNA Input Fluorescent Linear Amplification Kits (Agilent Technologies, Santa Clara, CA, USA), cDNA was reverse transcribed from each RNA sample, and cRNA was then transcribed and fluorescently labeled from each cDNA sample. The mouse heart tissue cRNA samples were labeled with Cy5 and the Universal Mouse reference cRNA was labeled with Cy3. The resulting cRNA was purified using an RNeasy kit (Qiagen, Valencia, CA, USA) followed by quantification of the cRNA by spectroscopy using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). 825 ng Cy3- and Cy5- labeled and amplified cRNA was mixed and fragmented according to the Agilent technology protocol.
Hybridization protocol
cRNA was hybridized to 4×44K whole human genome microarray slides from Agilent (Part G4112F) according to the manufacturer's instructions. The hybridization was carried in a rotating hybridization chamber in the dark at 65°C for 17 h.
Scan protocol
Slides were washed with Gene Expression Wash Buffer 1 and 2 (Agilent Technologies, Santa Clara, CA) followed by Acetonitrile. A final wash in Stabilization and Drying Solution was performed to prevent Cy-5 degradation by ozone. The array was scanned using an Agilent G2505B DNA microarray scanner under extended Dynamic range.
Data processing
The image files were extracted using the Agilent Feature Extraction software version 9.5.1 applying LOWESS background subtraction and dye-normalization.
