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

By Jun Zhang, Gregg Rokosh

This ebook provides either state-of-the-art and proven tools for learning cardiac gene expression. The protocols supply a template for good examine, 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 to the publication highlights tools that facilitate overexpression or cardiac-specific precise gene deletion.

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Extra resources for Cardiac Gene Expression: Methods and Protocols (Methods in Molecular Biology Vol 366)

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Do not allow the microarrays to dry. 3. Preparation of the Hybridization Cocktail and Hybridization 1. Prepare the hybridization cocktail for a single probe array hybridization as shown in Table 1. The recipe takes into account that it is necessary to make extra hybridization cocktail owing to a small loss in volume during each hybridization. Scale up the volumes for hybridization to multiple arrays. Add the cRNA last. If an error is made, you will not have to redo the fragmentation step. 2. Heat the hybridization cocktail to 99°C for 5 min prior to use.

Preparing the Bioanalyzer The electrodes should be decontaminated before each run. 1. Place a washing chip (electrode cleaner) filled with 350 µL RNaseZap in the Bioanalyzer and close the lid for 1 min. Remove the washing chip from the Bioanalyzer. 2. Place another washing chip filled with 350 µL DEPC-water in the Bioanalyzer, and close the lid for 30 s. 3. Remove the washing chip and leave the lid open for 10 s to allow the electrodes to dry. 3. Loading the Chip Remove the chip from packaging and inspect the under-side wafer of the chip for any defects.

The following steps are carried out at RT. Work quickly through the whole cleanup procedure to minimize the risk of RNA degradation. 22. Add 10 µL β-mercaptoethanol (β-ME) per 1 mL buffer RLT before use. Caution: β-ME is toxic; dispense in a fume hood and wear appropriate protective clothing. Buffer RLT is stable for 1 mo after addition of β-ME. 23. Add 350 µL Buffer RLT to the 100 µL RNA-containing solution, and mix thoroughly. 24. Add 250 µL ethanol (96–100%) to the diluted RNA, and mix thoroughly by pipeting.

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