How much of the first-strand cDNA reaction should I load for PCR?
While the volume is dependent on the starting amount of RNA used for the first-strand synthesis and the abundance of the target gene, we'd recommend starting with 10% of the first-strand reaction for your PCR reaction.
Can I use elevated temperatures for reverse transcription with Moloney Murine Leukemia Virus (M-MLV)?
When using GeneAmp RNA PCR kit (part number N808-0017) with M-MLV, the recommended temperature for reverse transcription is 42°C. It is not recommended that reverse transcription be done at a higher temperature with this enzyme because it will start to lose significant activity. If it is necessary to do reverse transcription at a higher temperature due to strong secondary structure or high G+C content, the recommended enzyme to use would be rTth DNA polymerase, which is included in the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR kit (part number N808-0069).