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TIGR Potato cDNA Microarray Protocols

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TIGR Potato cDNA Microarray Protocols

We have optimized hybridization and detection methods for use with the TIGR Potato cDNA Microarrays. We have compared several parameters including RNA isolation, mRNA labeling, hybridization, and scanning. The foundation of our methods are described in "A Concise Guide to cDNA Microarray Analysis-II" in Biotechniques (2000), 29(3):548-562 (Click here for the PDF file).

Two alternative RNA isolation methods are provided: one using phenol and one using trizol. We use a phenol method with carbohydrate-enriched tissue such as tubers and trizol for other tissues such as leaves.

We have provided a method to label cDNA as a robust way to test the hybridization procedure.

We have provided two methods for labeling RNA: direct labeling and amino allyl labeling. Both methods work fine, yet amino allyl requires only 20ug of total RNA.

We have provided a description of the techniques and software used for the array acquisition, analysis, "bad spot" filtering and normalization procedures.

Protocol for the TIGR file naming convention explains the terms used to store microarray data in the Solanaceae Gene Expression Database (SGED) and uniquely identify each hybridization parameters.

• 1. PCR Amplification Methods
• 1.1.1 PCR amplification of cDNA inserts
• 2. Array Printing Methods
• 2.1.1 Array Printing
• 3. RNA Isolation Methods
• 3.1.1 RNA isolation using Trizol
• 3.2.1 RNA isolation using phenol
• 3.3.1 RNA isolation using Qiagen RNAeasy kit
• 4. cDNA Probe Labeling Methods
• 4.1.1 Labeling of cDNA inserts using PCR
• 5. RNA Labeling Methods
• 5.1.1 Direct labeling of RNA
• 5.2.1 Indirect labeling of RNA
• 5.2.2 Indirect labeling of RNA
• 6. Hybridization Methods
• 6.1.1 Hybridization using directly labeled probes
• 6.2.1 Hybridization using indirectly labeled probes
• 6.2.2 Hybridization using indirectly labeled probes
• 6.2.3 Hybridization using indirectly labeled probes
• 6.2.4 Hybridization using indirectly labeled probes
• 7. Quantification and Normalization Methods
• 7.1.1 Quantifying and normalizing the data using LOWESS
• 8. Data processing
• 8.1.2 Microarray Files and Directories Naming Convention and Data Distribution
• 9. mRNA Amplification and Hybridization
• 9.1.1 mRNA Amplification and Hybridization

 

Last modified: Monday, 16-Mar-2009 11:06:14 EDT

    

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