strain: C57BL/6J gender: male age: 8week tissue: heart
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted individually using the RNeasy Mini Kit for liver tissue and RNeasy Fibrous Tissue Mini Kit for myocardium (QIAGEN, Chatsworth, CA), according to the manufacturer’s protocol. RNA was analyzed to determine if it met the minimum purity and integrity standards for total RNA quality (i.e., OD 260/280>1.8, OD 260/230>2.0, and minimal degradation as determined using a NanoDrop 1000A spectrophotometer; NanoDrop Technologies, Wilmington, DE). The quality and degradation of the isolated RNA were estimated after electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Heart or liver samples were pooled in each group at the indicated time-points and amplified using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion, Austin, TX), as recommended by the manufacturer.
Label
Cy5
Label protocol
Dissolve aRNA sample in 5μl of 0.2M Sodium bicarbonate buffer(pH9.0). Add 5μl of CyDye (dissolved in 45μl of DMSO/vial ;Amersham Bioscience) to sample. Mix well by vortex. Incubate for 1hour at 40°C keep in dark.
strain: C57BL/6J gender: male age: 8week tissue: heart
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted individually using the RNeasy Mini Kit for liver tissue and RNeasy Fibrous Tissue Mini Kit for myocardium (QIAGEN, Chatsworth, CA), according to the manufacturer’s protocol. RNA was analyzed to determine if it met the minimum purity and integrity standards for total RNA quality (i.e., OD 260/280>1.8, OD 260/230>2.0, and minimal degradation as determined using a NanoDrop 1000A spectrophotometer; NanoDrop Technologies, Wilmington, DE). The quality and degradation of the isolated RNA were estimated after electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Heart or liver samples were pooled in each group at the indicated time-points and amplified using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion, Austin, TX), as recommended by the manufacturer.
Label
Cy3
Label protocol
Dissolve aRNA sample in 5μl of 0.2M Sodium bicarbonate buffer(pH9.0). Add 5μl of CyDye (dissolved in 45μl of DMSO/vial ;Amersham Bioscience) to sample. Mix well by vortex. Incubate for 1hour at 40°C keep in dark.
Hybridization protocol
Incubate at 50°C for 20 hour in a humid chamber.
Scan protocol
The microarrays were then scanned using ScanArray GX (PerkinElmer, Wellesley, MA) and signal values were calculated using GenePix Pro 4.0 software (Axon Instruments, Union City, CA). Image output files were examined visually for major defects and hybridization artifacts.
Description
Liver or heart samples were amplified individually without mixing. As a reference for hybridization, we used mixed RNA samples from the sham group at various time-points. Test and reference samples were labeled with fluorescent Cy5 and Cy3, respectively, mixed, and hybridized on a microarray. Supplementary txt file: our nomalized values are in column AI. But, if signal intensity of Cy3 or Cy5 is less than 100, the column AI is blank. In this situation, normalized values is in column AL or AO.
Data processing
The signal intensity of each spot was corrected by subtracting adjacent background signals. To normalize the data, we averaged the intensities of all spots obtained with Cy3 and Cy5 in each of the 48 rectangles, and adjusted the intensity of each corrected DNA spot by the average intensity ratio of Cy5:Cy3 (equal to 1.0). This global normalization of intensity provided a smaller variance of the Cy5:Cy3 ratio and results similar to those obtained via normalization using housekeeping genes.
