Running the preloaded TaqMan Array is a simple, two-step process. During the first step, total RNA (nonenriched, containing small RNA species; e.g., isolated with the mirVana™ miRNA Isolation Kit) is reverse transcribed using predefined RT primers that are specific for only the mature miRNA species. This step has been reduced to eight independent RT reactions using the Multiplex RT pools (see MicroRNA Profiling: Save Time and Sample Using Real-Time PCR), resulting in significant time and sample savings. For this step, microarrays require 1000 ng total RNA, and individual assays require 366 ng total RNA. The Multiplex RT pools allow you to use as little as 80 ng or 10 ng/RT reaction. In the second step, each of the resulting eight RT pools containing cDNA template are diluted, mixed together with TaqMan Universal PCR Master Mix, and loaded into each of the eight fill ports on the TaqMan Array (Figure 1). The card is briefly centrifuged to distribute samples to multiple wells on the array, and then sealed. Within minutes the real-time PCR can be run using the Applied Biosystems 7900HT Fast Real-Time PCR platform. Standard 7900RQ software is used for data analysis.
Fast, Sensitive miRNA Profiling by Real-Time RT-PCR.