T7Eberwine Type Amplification

The most popular methods of RNA amplification for microarray-based transcription profiling are modified from the classic Eberwine protocol (34). The basic premise of this approach is that mRNA is converted into cDNA using an oligo-dT primer that has an additional sequence encoding the T7-promoter appended to it. After performing a second strand cDNA synthesis, an artificial gene under the control of a T7-promoter is created. This gene can then be transcribed by T7-polymerase creating multiple RNA copies of each gene.

This basic technique is utilized by many researchers and is a standard part of the Affymetrix, Agilent, and Illumina labeling protocols. Proponents of this technique point to the fact that it is linear in nature, while providing great increases in sensitivity. This protocol can be performed in tandem (multiple rounds of amplification) to obtain even greater amounts of material; however, each successive round of amplification does introduce more bias (35). With multiple rounds of amplification, as little as 10 ng of total RNA can be used to create labeled material for microarray analysis (35, 36). While this thousand-fold increase in sensitivity is impressive, the requirement for a minimum of 1000 cells can still be problematic for many researchers using LCM techniques for tumor profiling. Another drawback of this method is the time requirement for amplification: a single round of amplification adds between 1.5 and 2 days of time to the labeling procedure, whereas two rounds of amplification can add 3 days (37).

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