A variety of factors that influence real-time reverse transcriptase (RT)-PCR results were examined. Real-time reaction volumes can be decreased from the manufacturers suggested volumes (50µl) to as little as 5µl. Errors introduced through volume handlings can be significantly mitigated through the use of electronic pipettes. Random hexamers are compared against the use of a specific reverse transcription primer. Negative effects of non-target total RNA on the real-time RT-PCR results are detailed. Finally the amount of the RT reaction that can be successfully loaded into the real-time PCR reaction was determined and results indicate that a considerable amount can be successfully utilized. It was determined that the RT reaction buffer alone impacts on the quality of the real-time PCR results. The results demonstrate simple and effective methods to increase the resolution and reliability of the real-time RT PCR protocols.
