By Yurong Liang, Xin Lu, David L. Perkins (auth.), Jun Zhang, Gregg Rokosh (eds.)
Cardiac Gene Expression: equipment and Protocols provides either state-of-the-art and tested equipment for learning cardiac gene expression. The protocols offer a template for sturdy examine, and canopy the method via screening, research, characterization, and sensible affirmation of novel genes or identified genes with a brand new function.
Section I, Cardiac Gene Expression Profiling: the worldwide point of view, discusses a number of diverse techniques to studying, picking out, and examining alterations in transcriptome gene expression. part II, Cardiac Gene rules: 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 legislation mechanisms by way of targeting the interplay of transcription elements with their cognate cis binding components. part IV, In Silico evaluation of Regulatory cis-Elements and Gene rules, and part V, Cardiac unmarried community Polymorphisms, emphasize new analytical techniques for interpreting the sensible 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 tools that facilitate overexpression or cardiac-specific certain gene deletion.
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Extra info for Cardiac Gene Expression: Methods and Protocols
Prechill 100 mL PBS and a 75-mm tube with TRIZOL reagent on ice. 2. Weigh the tissue while taking care to keep it frozen (see Note 3). An adult mouse heart weighs about 100 to 170 mg. Homogenize tissue samples in 1 mL of TRIZOL reagent per 50 to 100 mg of tissue using a power homogenizer with a sawtooth generator probe (see Note 4). The sample volume should not exceed 10% of the volume of TRIZOL reagent used for homogenization. 3. Transfer samples into 75-mm tubes filled with the appropriate amount of room temperature (RT) TRIZOL reagent using a prechilled spatula (see Note 5).
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. 4. Insert the carriers into the preheated 45°C oven. Make sure that the carriers are properly secured.
5-mL Collection Tube (supplied), and pipet 11 µL of RNase-free water directly onto the spin column membrane. Centrifuge for 1 min at maximum speed (≤25,000g). 10. Determine the cRNA yield by measuring absorbance at 260 nm, and determine the purity by measuring the 260:280 nm ratio using a spectrophotometer or the Nanodrop ND-1000. 11. ) (see Note 14). 6. Fragmentation of the Biotin-Labeled cRNA To achieve optimal binding sensitivity on Affymetrix GeneChip probe arrays, the biotin-labeled cRNA is fragmented by metal-induced hydrolysis into 35 to 200 base fragments (see Note 15).