Printing Methods

Contents Introduction ............................................................................................................ 159

Microarray Technologies .............................................................................. 159 List of Protocols ............................................................................................ 162

Protocol 10.1: Micro Spotting Pin Selection Performance Optimization ...................................................................................................... 163

Protocol 10.2: Surface Chemistry .......................................................................... 164 Protocol 10.3: Practical Considerations for Optimizing Protein

Microarray Manufacturing ............................................................................ 166 Protocol 10.3.1: Experimental Design .......................................................... 166 Protocol 10.3.2: Selecting Peptides/Proteins ................................................ 167 Protocol 10.3.3: Sample Preparation ............................................................ 167 Protocol 10.3.4: Setting up the Source Plates for Printing ........................... 168 Protocol 10.3.5: Executing the Print Run ..................................................... 169

How It Works .................................................................................... 169 Required Materials ............................................................................ 169 Step-by-Step Protocol ....................................................................... 169 Troubleshooting Guide ..................................................................... 178 Microarray Terms Used .................................................................... 181

Protocol 10.4: Processing Protein (Serum-Based) Microarrays ............................ 182 How It Works ................................................................................................ 182 Required Materials ........................................................................................ 182 Reagent Preparation ...................................................................................... 183 Step-by-Step Protocol ................................................................................... 183 Key References ............................................................................................. 184 Troubleshooting Guide ................................................................................. 184

Note 1: Track Microarrays ................................................................ 184 Note 2: Wash/Block Processing ........................................................ 184 Note 3: Incubation of Serum Test Sample ........................................ 184 Note 4: First Wash ............................................................................ 184

Note 5: Incubate Labeled Antibody .................................................. 185 Note 6: Dry Microarrays ................................................................... 185

Protocol 10.5: Ex Situ DNA Microarray Manufacturing and Processing .............. 185 Protocol 10.5.1: Experimental Design .......................................................... 185 Protocol 10.5.2: Sample Preparation ............................................................ 186 Protocol 10.5.3: Setting up the Source Plates for Printing ........................... 186 Protocol 10.5.4: Executing the Print Run ..................................................... 186 Protocol 10.5.5: Processing the DNA Microarray for Gene

Expression .................................................................................................186 Microarray Workflow ........................................................................ 186 Amplification .................................................................................... 187 Labeling ............................................................................................ 187 Prehybridization ................................................................................ 188 Hybridization .................................................................................... 188 Stringency Wash ............................................................................... 189 Microarray Scan Output ................................................................... 189

Protocol 10.6: The H25K Master Protocol ............................................................ 190 How It Works ................................................................................................ 190 Protocol 10.6.1: RNA Isolation from Tissue ................................................ 190 Protocol 10.6.2: RNeasy MinElute Cleanup ................................................. 190 Protocol 10.6.3: cDNA/senseRNA Preparation ............................................ 191

Protocol 10.6.3.1: SenseAMP Procedure for First Strand cDNA Synthesis ............................................................................ 191

Protocol 10.6.3.2: Purification of cDNA .......................................... 191 Protocol 10.6.3.3: Tailing of First Strand cDNA .............................. 192 Protocol 10.6.3.4: T7 Promoter Synthesis ........................................ 192 Protocol 10.6.3.5: In Vitro Transcription .......................................... 193 Protocol 10.6.3.6: Purification of senseRNA .................................... 193 Protocol 10.6.3.7: Quantitation of senseRNA .................................. 194

Protocol 10.6.4: cDNA Synthesis and Indirect Aminoallyl Fluorescent Labeling Kit ...................................................................................... 194 How It Works .................................................................................... 194 Required Materials ............................................................................ 195 Step-by-Step Protocol ....................................................................... 195

Protocol 10.6.5: H25K Hybridization of Labeled cDNA ............................. 197 Troubleshooting Guide ................................................................................. 198

Note 1: Hybridization Buffer ............................................................ 198 Note 2: Contamination Issues ........................................................... 198 Note 3: Gasket .................................................................................. 198 Note 4: Humidity Control ................................................................. 199 Note 5: Coverslip Directions ............................................................ 199 Note 6: Hybidization Time ............................................................... 199 Note 7: Directions for Using the Hybridization Cassette ................. 199 Note 8: Removal of Coverslip ..........................................................200