Many researchers are currently pursuing the development of unbiased, accurate methods to measure the whole proteome or the whole transcriptome in small biological samples at or near the single-cell level in order to effectively identify unique biomarkers of disease. In the work described here, whole transcriptome amplification of Jurkat T-cell mRNA was achieved using a 2-step linear amplification method with the first step being the Eberwine amplication method performed in a packed bed on a microfluidic chip. The Eberwine amplification method works without themocycling or bias and gives ~1000-fold increase in concentration in benchtop experiments, though it requires relatively high starting concentrations. For this amplification step, microfluidics offer the great advantage of dramatically increasing the effective concentration of the original starting material through immobilization of the cDNA library onto microbeads packed in a ~1 nL reaction volume. The second amplification step is performed off-chip, and results indicate that we are able to raise the sample concentration enough for use on microarrays.

The reaction was performed in a device made by multilayer soft-lithography techniques with hydraulic valves that close partially to retain the cDNA-functionalized microbeads under constant flow. Specific high and moderate-copy genes were detected post-amplification using off-chip RT-PCR to confirm amplification. RNA length (i.e. amplified sample quality) was measured using capillary electrophoresis and found to match the average template size (1 kb), indicating little or no bias was introduced in the sample that would lead to false gene expression profiling.