Fig. 1

HyCEAD workflow. Integration of the hybridization of reverse transcription primers with purification of poly-A + RNA from crude lysates on magnetic beads removes the need for RNA extraction prior to processing. A large number of reverse primers at high concentrations in the lysate provides favorable hybridization kinetics, and unhybridized primers are washed away along with unbound nucleic acids and other sample components before reverse transcription. This essentially eliminates the interaction of unbound reverse primers during reverse transcription, thereby greatly minimizing the production of primer dimers and off-target artifacts. Although many different gene-specific forward primers are present in solution during PCR amplification, the concentration of the gene-specific primers is relatively low (3.4 nM) to minimize primer-dimer formation, and the universal primers are present at 500-fold greater concentrations. Although primer dimers nevertheless may form between different forward gene-specific primers, they will have the forward universal primer at one end and the complement of the forward universal primer at the other end. As a result, they will form stable hairpin structures which will not be efficiently amplified [30]. These inherent features of the HyCEAD process [28] as well as the design of the gene-specific forward primer (see discussion of primer design above) minimizes the potential interference of primer dimers in the multiplex amplification. The specificity of the process is determined by four levels of selection: capture of poly-A+ RNA, target amplification with two gene-specific primers per target and detection of specific biotinylated amplicons by Ziplex probes that hybridize near the center of the amplicons