I am trying to optimise a real-time RT-PCR on FIV RNA. I have been using SYBR green and got the real-time PCR working well as a 2-step process using high fidelity reverse transcriptase in the first step. When I try to run the same RNA samples through as a 1-step process (using the same enzyme), there are no amplification products. When I use cDNA as the template (with the RT also in the mixture), it still doesn't work, even though the cDNA produced good amplification in the 2-step process. I can't work out why this is occurring - could the RT somehow be interfering with the SYBR green in my reaction???
Any help would be appreciated!