Question

Outline the differences between RT-PCR and qPCR and what purpose would you use qPCR.

Answer 1

Difference between RT- PCR and qPCR:

-RT-PCR is used to amplify reverse transcription of DNA code where as qPCR measures the amplification.

-RT-PCR is used for amplification where as qPCR is used for quantification.

- qPCR is quantitative nature, while RT-PCR is not.

-RT-PCR is used for the detection of cancer and diagnosis of various genetic diseases where as qPCR is used to identify various viral infections.

- qPCR is more expensive than RT-PCR.

-RT-PCR is more difficult set than qPCR

-RT-PCR, starting genetic material is RNA, where it is transcribed to DNA complement by reverse transcriptase enzyme, where as in qPCR they have a qPCR machine and fluorescent dye in PCR reaction.

-in RT-PCR gel electrophoresis is required where as in qPCR, there is no need to do gel electrophoresis to see the amplified PCR product.

Purpose of qPCR:

qPCR is used to detect, quantify and characterize nucleic acid for numerous applications. In qPCR fluorescent labelling helps in the collection of data as PCR progress, this technique has many benefits due to range of methods available.

In dye based qPCR fluorescent labelling allows the quantification of amplified DNA molecule. In each cycle fluorescence is measured. Fluorescence signal increases proportionally to the amount of DNA replicated ,so DNA is quantified in real time. Disadvantage-only one target can be examined at a time.

Ine probe based qPCR, many targets can be detected simultaneously in each sample. But it requires optimisation and design of a target specific probe. There are several types of probes available, but the most common type is hydrolysis probe. The fluorescence signal from probe based qPCR is proportional to the amount of the probe target sequence. Since probe based qPCR is more specific than dye based qPCR, it is more often used in qPCR diagnostic assays.