( Optimizing specific...)Simple Approaches for Optimization of RT-PCR

Optimizing specificity

Optimal specificity may be achieved by using the optimal annealing temperature for a given pair of primers in the PCR step of RT-PCR. The optimal annealing temperature for a pair of primers may be determined as follows. First, estimate the primer melting temperature (T_m) for each primer based on the number of G, C, A, and T residues in each primer, according to the following equation: T_m = [(G+C) x 4C] + [(A+T) x 2C]. Second, conduct RT-PCR with the pair of primers using an annealing temperature that is well below the lower Tm for the two primers (5 to 10C lower, for example). Third, increase the primer annealing temperature in increments of 2 to 3C in subsequent or parallel RT-PCR experiments, until optimal specificity is achieved. For best results, use pairs of primers that are well matched in terms of their T^_m values.

Specificity may also be improved by use of relatively low amounts of primers in RT-PCR reactions. For relatively long (≥1.5 kb) RT-PCR products in particular, decreasing the concentration of each primer from 0.4μM to 0.075μM can have a positive effect on specificity (Fig. 4). Note that this approach tends not to work as well with relatively short (0.5 kb) RT-PCR products. In such cases low primer concentrations may result in decreased sensitivity and yield (Fig. 3).

Optimizing yield

Excellent yields may be obtained by optimizing factors such as product length (shorter products may result in higher yields), RNA concentration (more RNA may result in higher yields), and MgCl2 concentration (1.5mM to 2.5mM typically works well in one-step RT-PCR). One additional approach, which works well for some targets, is to increase the amount of RT-PCR enzyme mix in the reaction. For some relatively long (≥1.5 kb) RT-PCR targets, doubling t
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