The major difference between real-time PCR and reverse transcriptase PCR (RT-PCR) is the target molecule and the detection method.

Real-time PCR can be used to detect and quantify both DNA and RNA, while RT-PCR is specifically used to detect and quantify RNA. This is because RT-PCR includes an additional step called reverse transcription, which converts RNA into complementary DNA (cDNA) before it is amplified by PCR.

Real-time PCR also uses a different detection method than RT-PCR. In real-time PCR, the amplified DNA is detected using a fluorescent probe. The amount of fluorescence is proportional to the amount of amplified DNA, which allows for real-time quantification of the target molecule. In contrast, RT-PCR typically uses agarose gel electrophoresis to detect the amplified cDNA. This method is less sensitive than real-time PCR and does not provide real-time quantification.