Realtime RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, can quantify an RNA of interest as the reaction progresses.

To begin, reverse transcriptase copies the RNA into complementary, or cDNA. RNase H  digests the original RNA, leaving behind small primers attached to the cDNA.

A complementary strand is then synthesized by DNA polymerase. 

Targeted amplification can then be carried out using PCR to create exponential copies of specific segments. The two strands are separated at the start of each cycle at high temperatures. Complementary oligonucleotide primers then anneal to each cDNA strand, and are extended by DNA polymerase.

The DNA can be quantified using one of two different fluorescence-based detection methods. One method uses dyes that only fluoresce when bound to double-stranded DNA.

The dye binds to DNA, where the original strand is paired with a newly synthesized complementary strand. At the end of each PCR cycle, an appropriate wavelength of light excites the bound dye.

The other method uses complementary sequence-specific oligonucleotide probes linked to a fluorophore and a quencher molecule.

The reaction is continuously exposed to an appropriate wavelength of light, and the quencher molecule absorbs the fluorophore’s fluorescence when near each other. DNA polymerase detaches the fluorophore during extension, preventing the quenching.

The probe-based method is specific, as it only attaches to the target sequence. In contrast, the dye-based method is non-specific and binds to all double-stranded DNA.

In either case, fluorescence emitted by the excited fluorophore is sensed by photodetectors that convert the signals received to a digital output.

The threshold cycle, or Ct,  is the number of PCR cycles for the fluorescence to reach a set level well above the background fluorescence.

The threshold cycle is inversely proportional to the amount of target RNA in the initial sample — the lower the threshold cycle, the higher the amount of the RNA of interest.

Absolute quantification compares the threshold cycle or fluorescence intensity to that of a standard curve prepared using known DNA concentrations.

Alternatively, relative quantification compares the fluorescence of a sample to that of a reference. This method can be used to compare changes in gene expression under different conditions.