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Microgenomic Expression Profiling

spectrophotometric readings. This novel formofmicro-spectrophotometry,inconcert with exisitng technologies commonly used for RNA quality assessment, allows for unprecedented process control during microgenomic experiments. The methods described herein are intended as general guidelines for microarray probe synthesis from limited amounts of RNA (≤2000 cells). Following laser microdissection of the desired cell population, RNA is isolated and then linearly amplified to generate antisense RNA (aRNA). When the starting cell population is very limited, a second round of linear amplification if necessary in order to have sufficient quantities of aRNA to use for probe synthesis. In the second round of amplification, complimentary DNA (cDNA)isgeneratedthroughfirstandsecond strand synthesis, and then used as template for amino-allyl dUTP incorporation during a second round of RNA amplification in preparation for fluorescent dye labeling. aRNA quality is typically assessed at critical steps in the workflow using the Agilent bioanalyzer system. Final probe quantity, as well as efficiency of dye incorporation, is determined using the NanoDrop ND1000 spectrophotometer in preparation for microarray analysis.

Instrumentation and Kits


Methods
Special Considerations:
◊ Minimize degradation by storing RNA at -70C.

◊ Frequent freeze-thaw cycles accelerate RNA degradation.

◊ RNase contamination will cause experimental failure. Adhere to the following recommendations to minimize RNase contamination:
Wear disposable gloves and change them frequently.
Clean work surfaces, instruments, racks, and reagent bottles with com mercially available RNase and DN asedecontaminationsolutionsbefore performing reactions.
After putting on
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