Realtime RT-PCR

Real-time RT-PCR uses reverse transcriptase to copy RNA into complementary DNA, or cDNA. RNase H then digests the original RNA, leaving small primers attached to the cDNA. DNA polymerase synthesizes a complementary strand, creating double-stranded DNA that can be amplified through PCR cycles for exponential copying of specific segments.

Dye-based detection uses fluorescent dyes that bind to all double-stranded DNA and fluoresce when excited by light at the end of each PCR cycle. Probe-based detection uses sequence-specific oligonucleotide probes linked to a fluorophore and quencher molecule. The probe-based method is specific to target sequences, while the dye-based method is non-specific, binding to any double-stranded DNA in the reaction.

The threshold cycle, or Ct, is the number of PCR cycles required for fluorescence to reach a set level above background. The Ct value is inversely proportional to the initial amount of target RNA—a lower Ct indicates more RNA was present in the original sample. This relationship allows researchers to quantify RNA levels by measuring when fluorescence becomes detectable.

In probe-based detection, the fluorophore is initially quenched by a nearby quencher molecule that absorbs its fluorescence. When DNA polymerase extends the complementary strand during PCR, it detaches the fluorophore from the probe. This separation prevents quenching, allowing the fluorophore to emit fluorescence that is detected by photodetectors and converted to a digital signal.

Absolute quantification compares the threshold cycle or fluorescence intensity of a sample to a standard curve prepared using known DNA concentrations. By plotting Ct values against known template amounts, researchers can determine the exact quantity of target RNA in unknown samples. This method requires generating standards with defined concentrations before analyzing experimental samples.

Relative quantification compares the fluorescence of a test sample to that of a reference sample rather than to a standard curve. This method measures changes in gene expression under different conditions without determining absolute RNA amounts. Relative quantification is useful for comparing expression levels between samples, such as treated versus untreated cells.

Real-time RT-PCR requires a specialized PCR machine equipped with optical detectors and light sources to enable real-time quantification during amplification. Traditional PCR machines lack these components and cannot measure fluorescence signals as the reaction progresses. The specialized equipment allows photodetectors to continuously monitor and convert fluorescence signals to digital output for quantitative analysis.