What can I do if a particular amplicon failed in a multiplex reaction using the LunaScript Multiplex One-Step RT-PCR Kit?

First, ensure that the amplicon length is within the recommended range of 100-1500 bp. If it is but fails to amplify in a multiplex reaction, perform a single-plex RT-PCR to evaluate the performance of the primers. Redesign the primers following guidelines in the General Tips and Consideration Section of the manual if no successful amplification was obtained in a single-plex RT-PCR. If the primer set works well in a single-plex RT-PCR, increase its concentration in the primer pool, (where each primer normally has the same concentration), by titration. This is likely to improve its amplification in a multiplex RT-PCR reaction. In addition to individual primer concentration rebalancing, as the number of targets increases in multiplex RT-PCR, the individual primer concentration may decrease, therefore, increasing the annealing time may also improve overall RT-PCR performance. For best results, we recommend empirically determining the annealing time in the context of primer concentration with the final total primer concentration between 1-4 µM.

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