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Real Time RT-PCR

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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.

Overview

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.

The real-time quantification of the number of amplified products is carried out either using fluorescent dyes or sequence-specific probes linked to a fluorophore. The fluorescent dyes used in this case can specifically bind to double-stranded DNA and exhibit fluorescence only when bound to DNA. Fluorescence is measured at the end of each PCR cycle after the synthesis of the complementary strand. In the case of fluorophore-labeled probes, fluorescence is exhibited when the fluorophore is cleaved from the probe during the synthesis of the complementary strand. Fluorescence exhibited by these molecules is then detected by photodetectors that convert the fluorescence signals to a readable format. Real-time RT-PCR requires a specialized PCR machine equipped with a detector to enable real-time quantification, as a traditional PCR machine is not equipped for such analysis.

The output can be analyzed in a number of ways. One way is to count the number of cycles required for the fluorescence to reach detectable levels. The threshold cycle, denoted by C_t, is when the fluorescence is detected over and above the background noise. The greater the quantity of the target in the starting material, the faster the significant increase in fluorescence will appear, resulting in a lower C_t.The C_t value finds application in the downstream quantification or detection of the presence or absence of the target sequence. Additionally, comparing the C_t values of samples of unknown concentration with a series of C_t values obtained from standard concentration can determine the amount of template in an unknown reaction. This is known as absolute quantification and can also be carried out by comparing the fluorescence to a standard curve prepared using known DNA concentrations.

Transcript

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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.

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