Hi Amit
terms are used losely and inter changeably in my opinion. Thus,
1. I take RT PCR to mean and END POINT assay in which cDNA is made; PCR performed and the amplicons run on an agarose gel. Cheap simple but non necessarily quantitative; especially if your amplification results in signal saturation so it becomes difficult to truly distinguish expression differences between 2 or more genes
2. In contrast, real time PCR as the name implies is an assay that allows you to monitor amplification efficiency - linked to starting copy number of expressed gene x - usually using sybr green and thus select a point in the amplification plot termed the ct value where amplification is still in exponential mode. By virtue of this you can accurately quantify 1 or more genes and thus look at absolute and relative expression differences. More expensive than gel based RT PCR but much more quantitative
Hi Amit
terms are used losely and inter changeably in my opinion. Thus,
1. I take RT PCR to mean and END POINT assay in which cDNA is made; PCR performed and the amplicons run on an agarose gel. Cheap simple but non necessarily quantitative; especially if your amplification results in signal saturation so it becomes difficult to truly distinguish expression differences between 2 or more genes
2. In contrast, real time PCR as the name implies is an assay that allows you to monitor amplification efficiency - linked to starting copy number of expressed gene x - usually using sybr green and thus select a point in the amplification plot termed the ct value where amplification is still in exponential mode. By virtue of this you can accurately quantify 1 or more genes and thus look at absolute and relative expression differences. More expensive than gel based RT PCR but much more quantitative
I think it's unfortunate that both reverse transcriptase and real-time begin with r t; this has led to confusion and using the terms interchangeably. Reverse transcriptase PCR came first and was abbreviated RT-PCR. My understanding of the original meaning of RT-PCR was that it used a reaction mixture that included RNA, reverse transcriptase, and your PCR primers; the first part of the thermal reaction was to make cDNA out of the RNA, and the second part was to make the amplicon. The results of the PCR could be measured either with an end-point gel or in real time. I think it is much more common now to separate these reactions; that is, reverse transcribe the RNA first and measure the quality of your cDNA and then go on to do a PCR. In contrast, real-time PCR can be done starting with RNA (as described above), cDNA, or gDNA, but it uses fluorescence to monitor the amplification efficiency in real time.
Dear Laurence Stuart Dawkins-Hall,
Thanks for your answer. Very informative reply.
Regards
Amit Sarder
Dear Dr. Deborah Fenner,
You have mentioned some crucial points that clearly explain the differences. Thank you so much.
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