By Yurong Liang, Xin Lu, David L. Perkins (auth.), Jun Zhang, Gregg Rokosh (eds.)
Cardiac Gene Expression: equipment and Protocols offers either state of the art and demonstrated equipment for learning cardiac gene expression. The protocols offer a template for good study, and canopy the method via screening, research, characterization, and practical affirmation of novel genes or identified genes with a brand new function.
Section I, Cardiac Gene Expression Profiling: the worldwide viewpoint, discusses numerous varied methods to analyzing, picking out, and studying alterations in transcriptome gene expression. part II, Cardiac Gene law: Gene-Specific mRNA size within the Myocardium, outlines extra delicate and gene-targeted expression tools. part III, Cardiac Gene law: Promoter Characterization within the Myocardium, offers protocols for the examine of underlying gene law mechanisms via targeting the interplay of transcription elements with their cognate cis binding components. part IV, In Silico overview of Regulatory cis-Elements and Gene rules, and part V, Cardiac unmarried community Polymorphisms, emphasize new analytical techniques for decoding the useful components buried within the three billion nucleotides of the human genome and different version genomes. The concluding part, Gene Overexpression and focusing on within the Myocardium, highlights equipment that facilitate overexpression or cardiac-specific special gene deletion.
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7. Do not leave the samples on ice for a prolonged period. Samples may be stored at –20°C for later use. 3. just below to avoid unnecessary freeze/thaw cycles that may impact the integrity of the cDNA. 3. Cleanup of Double-Stranded cDNA The components needed for cleaning up the cDNA are supplied with the GeneChip Sample Cleanup Module. Add 24 mL of 96 to 100% ethanol to the cDNA Wash Buffer prior to the first use. 1. Add 600 µL of cDNA Binding Buffer to the second strand synthesis reaction and mix by vortexing.
Preparing the Affymetrix GeneChip Array 1. , scratches on glass or wafer). Record the array type, lot number, and expiration date. 2. Wet the array by filling it through one of the septa with 200 µL of 1X Hybridization Buffer. 5 mg/mL 1X 10% 3 3 150 30 56 38 300 cartridge: one for filling and the second to allow venting of air from the hybridization chamber. Use 100 µL for Test3 arrays and 300 µL for a standard GeneChip format. 3. Insert the array into the GeneChip cartridge carrier. Prepare a balanced carrier.
11. If there are no more samples to run for the day, shut down the Fluidics station following the procedure outlined in the Affymetrix User Manual for Eukaryotic Sample and Array Processing. 2. Scanning of Affymetrix GeneChip Probe Arrays The scanner is also controlled by the Affymetrix Microarray Suite or GCOS. 1. Turn on the scanner at least 10 min before use to make sure that the laser is warmed up. 2. If the probe array was stored at 4°C, let it warm to room temperature before scanning. 3. Check that there are no bubbles visible in the glass window of the probe array.
Cardiac Gene Expression: Methods and Protocols by Yurong Liang, Xin Lu, David L. Perkins (auth.), Jun Zhang, Gregg Rokosh (eds.)