What is the difference between one-step and two-step RT-PCR?
One-step RT-PCR allows easier processing of large numbers of samples and helps minimize carry-over contamination since tubes are not opened between cDNA synthesis and amplification. By amplifying the entire cDNA sample, one-step RT-PCR can provide greater sensitivity-down to 0.01 pg total RNA. Only gene-specific primers can be used with one-step kits.
Two-step RT-PCR is useful for detecting multiple messages from a single RNA sample. You have greater flexibility to choose polymerase and primers than you do with one-step RT-PCR systems. When performing two-step RT-PCR you have the option of using either oligo(dT), random hexamers, or gene-specific primers, and then performing PCR in combination with either Platinum Taq DNA Polymerase, Platinum Taq DNA Polymerase High Fidelity, or your choice of other PCR enzymes.
Two-step RT-PCR is useful for detecting multiple messages from a single RNA sample. You have greater flexibility to choose polymerase and primers than you do with one-step RT-PCR systems. When performing two-step RT-PCR you have the option of using either oligo(dT), random hexamers, or gene-specific primers, and then performing PCR in combination with either Platinum Taq DNA Polymerase, Platinum Taq DNA Polymerase High Fidelity, or your choice of other PCR enzymes.