Guidelines for PCR Optimization using Phusion RT-PCR Kit
A separate RNA denaturation step is recommended. The denaturation step 5 minutes at 65°C should be performed before adding the RT Buffer and reverse transcriptase to the reaction.
- Primer Extension
The incubation for 10 minutes at 25°C extends oligo(dT) and random primers before the actual cDNA synthesis. Without the incubation at 25°C, the primers may dissociate from the template when the temperature is increased. When using gene-specific primers, this extension step is not necessary.
- CDNA synthesis
Incubation at 40°C will work for most templates, but the incubation temperature can be optimized between 37-48°C, if necessary. Increasing the temperature can be helpful if the template has strong secondary structures. A higher temperature can also improve specificity if gene-specific primers are used. Incubation about 48°C is not recommended.
In most cases, incubation for 30 minutes is sufficient. If the target is located near the 5´ end of a long transcript and olgio(dT) priming is used, or if the target is rare, cDNA synthesis time can be extended up to 60 minutes.
- Reaction Termination
The termination step at 85°C inactivates the M-MuLV RT. This prevents the reverse transcriptase from inhibiting the subsequent PCR reaction. Note: A separate RNase H treatment is not required. The cDNA can be directly used as a template in the subsequent PCR or stored at -20°C, if not used immediately.
- Initial Denaturation
Initial denaturation should be done at 98°C, although even higher temperatures can be used. 30 seconds at 98°C is recommended for most templates. This step can be extended up to 3 minutes, if necessary.
Keep the denaturation step as short as possible. 10 seconds at 98°C is adequate for most templates.
- Primer annealing
Note that the optimal annealing temperature for Phusion Hot Start II DNA Polymerase may differ significantly from that of Taq-based polymerases. Always use the Tm calculator and instructions on Finnzymes’ website to determine the Tm values of primers and optimal annealing temperature. For primers >20 nt, anneal for 5 seconds at a Tm +3°C of the lower Tm primer. For primers ≤20 nt, use an annealing temperature equal to the Tm of the lower Tm primer. If necessary, use a temperature gradient to find the optimal annealing temperature for each template-primer pair combination. The annealing gradient should extend up to the extension temperature (two-step PCR). Two-step cycling without an annealing step is recommended for high Tm primer pairs.
Extension should be performed at 72°C for cDNA templates, 40 seconds per 1 kb is recommended. However, many cDNA amplicons can be successfully amplified using even shorter extension times (15-30 seconds per 1 kb).
- Number of Cycles
The number of cycles required is dependent on the abundance of the original target RNA. Usually 25 cycles in PCR is adequate. If the target RNA is rare or if only a small amount of starting material is available, it may be helpful to increase the number of cycles to 35-40.