What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR?

PCR (polymerase chain reaction) is a widely used molecular biology technique that’s used for the amplification and detection of DNA and RNA sequences. RT-PCR, qPCR, and R-qPCR are all variations of the basic PCR technique. 

PCR

The traditional PCR technique is the simplest. The starting genetic material in this reaction is double-stranded DNA.

During this process double-stranded DNA is heat denatured in the first step. In the second step, primers align to the single DNA strands. In the final step, the primers are extended by DNA polymerase, resulting in two copies of the original DNA strand. The main applications of PCR include genetic testing and detection of pathogenic DNA.

RT-PCR

In RT-PCR (reverse transcription PCR), RNA is used as the starting genetic material. The process is almost similar to PCR but has an additional step that allows the detection and amplification of RNA.

During the RT-PCR process, the RNA is first reverse transcribed into a single-stranded copy of cDNA (complementary DNA) using the reverse transcriptase enzyme. The newly generated cDNA containing the reversed transcription is amplified by a DNA polymerase using the standard PCR. As a result double-stranded cDNA is produced.

qPCR

qPCR stands for Quantitative PCR. It involves PCR amplification of DNA in real time. The amount of DNA after each cycle of replication is measured using a fluorescent probe such as an interacting dye or a hydrolysis-based probe.

qPCR is typically used to determine the copy number of a DNA sequence of interest and to detect, characterize, and detect the presence of pathogens in a sample. 

RT-qPCR

RT-qPCR refers to reverse transcription quantitative real-time PCR. This technique combines RT-PCR with qPCR to facilitate the measurement of RNA levels through the use of cDNA in a qPCR reaction. This allows for rapid detection of changes in gene expression.

In this process, RNA transcripts are first quantified by reverse transcribing them into cDNA. This is followed by qPCR.