Cardiac Gene Expression: Methods and Protocols (Methods in by Jun Zhang, Gregg Rokosh

By Jun Zhang, Gregg Rokosh

This booklet offers either state of the art and confirmed tools for learning cardiac gene expression. The protocols offer a template for reliable learn, and canopy the method via screening, research, characterization, and useful affirmation of novel genes or identified genes with a brand new functionality. The concluding component of the booklet highlights tools that facilitate overexpression or cardiac-specific exact gene deletion.

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2. To minimize secondary structure, heat denature (70°C, 2 min) the samples before loading on the chip. 3. Add 1 µL of sample into each well. Empty wells should be filled with 6 µL water or the Nano marker. Accurate pipeting is very important. Use properly calibrated pipets. You may pipet up and down gently to mix samples in the wells. 4. Pipet 1 µL of the ladder into the ladder well. 5. Vortex the chip for 1 min at about 512g. Use a piece of tape or an elastic band to secure the chip in the adapter.

Set up and prime the Fluidics Station according to the Affymetrix User Manual for Eukaryotic Sample and Array Processing or the User’s Guides of the instrument. 3. Prepare the streptavidin phycoerythrin (SAPE) stain solution (Table 2). The table lists the components needed for one to five probe arrays, adding a small volume for pipeting losses. Always prepare the stain solution fresh on the day of use, and prepare enough solution for all arrays to be processed on that day. SAPE should be stored in the dark at 4°C.

3. (see Note 12), 4 µL 10X IVT Labeling Buffer, 12 µL IVT Labeling NTP Mix, 4 µL IVT Labeling Enzyme Mix, 8 µL RNase-free water. The total volume is 40 µL. 2. Mix well and incubate at 37°C for 16 h in a thermal cycler with heated lid to avoid condensation. 3. Labeled cRNA can be stored at –20°C for short-term storage or at –70°C for longer periods until further purified. 5. just below after the 16-h incubation to avoid freeze/ thaw cycles. 5. Cleanup and Quantification of Biotin-Labeled cRNA To determine the concentration of the cRNA accurately, it is necessary to remove unincorporated NTPs.

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