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Designing a Successful qRT-PCR Experiment

adjusted according to target abundance. By performing a primer titration, an optimal final primer concentration for a given target can be found. This can vary between 150 to 500 nM. An initial concentration of 400 nM for each primer is a useful starting point. The optimal TaqMan probe concentration can also vary according to relative target abundance, though 80 nM is generally a good starting point. We recommend a titration of 50 to 800 nM probe for optimization.

RNA Sample: The quality of the starting RNA template plays an important role in RT-PCR results. Ambion recommends RNAqueous-4PCR to obtain RNA free of DNA. An RNA sample containing contaminating DNA can be treated with Ambion's DNA-free Reagents to help eliminate genomic DNA contamination. To synthesize large amounts of RNA for a standard curve, Ambion recommends the MEGAscript High Yield Transcription Kit.

Control Reactions: Always include at least two controls in your experiments. The first controls for genomic DNA contamination by excluding RT from the reaction (minus RT control). The second, which contains all master mix components except the input RNA, controls for contamination of reagents and the reaction tubes (no-template control). Any other deviations from the master mix
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