Quantitative reverse transcription polymerase chain reaction, also called RT-qPCR, is used to detect and quantify RNA. Total RNA or mRNA is first transcribed into complementary DNA (cDNA). The cDNA is then used as the template for the quantitative PCR or real-time PCR reaction (qPCR). In qPCR, the amount of amplification product is measured in each PCR cycle using fluorescence. RT-qPCR is used in a variety of applications including gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research.
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RT-qPCR can be performed in a one-step or a two-step assay (Figure 1, Table 1). One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. One-step RT-qPCR only utilizes sequence-specific primers. In two-step assays, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies.
Figure 1. One-step vs. two-step RT-qPCR.
Learn more: One-step vs. two-step RT-qPCR
When designing a RT-qPCR assay it is important to decide whether to use total RNA or mRNA as the template for reverse transcription. mRNA may provide slightly more sensitivity, but total RNA is often used because it has important advantages over mRNA as a starting material. First, fewer purification steps are required, which helps ensure a more quantitative recovery of the template and a better ability to normalize the results to the starting number of cells. Second, by avoiding any mRNA enrichment steps, one can avoid the possibility of skewed results due to different recovery yields for different mRNAs. Taken together, total RNA is more suitable to use in most cases since relative quantification of the targets is more important for most applications than the absolute sensitivity of detection [1].
Explore: Total RNA isolation
To initiate reverse transcription, a short DNA oligonucleotide called a primer is required to anneal to the template RNA strand and provide reverse transcriptase a starting point for synthesis. Four different approaches can be used for priming cDNA reactions in two-step assays: oligo(dT) primers, random primers, or sequence specific primers (Figure 2 and Table 2). Often, a mixture of oligo(dT)s and random primers is used. Combining random primers and anchored oligo(dT) primers to diminish the generation of truncated cDNAs can help improve the reverse transcription efficiency and qPCR sensitivity.
Figure 2. Four different priming methods for the reverse transcription step in two-step assays of RT-qPCR.
Explore: Primers for reverse transcription
Reverse transcriptase (RT) is the enzyme that makes DNA from RNA. Some reverse transcriptases have RNase activity to degrade the RNA strand in the RNA-DNA hybrid after transcription. If an enzyme does not possess RNase activity, an RNase H may be added for better qPCR efficiency. Commonly used enzymes include Moloney murine leukemia virus reverse transcriptase and Avian myeloblastosis virus reverse transcriptase. For RT-qPCR, it is ideal to choose a reverse transcriptase with high thermal stability, because this allows cDNA synthesis to be performed at higher temperatures, helping ensure successful transcription of RNA with high levels of secondary structure, while maintaining their full activity throughout the reaction to help produce higher cDNA yields.
Explore: Reverse transcription enzymes
RNase H activity degrades RNA from RNA-DNA duplexes to enable efficient synthesis of double-stranded DNA. However, with long mRNA templates, RNA may be degraded prematurely which can result in truncated cDNA. Hence, it is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in qPCR applications because they can enhance the melting of RNA-DNA duplex during the first cycles of PCR (Figure 3).
Figure 3. RNase H activity of reverse transcriptases.
Learn more: Reverse transcriptase properties
PCR primers for the qPCR step of RT-qPCR should ideally be designed to span an exon-exon junction, with one of the amplification primers potentially spanning the actual exon-intron boundary (Figure 4). This design can help reduce the risk of false positives from amplification of any contaminating genomic DNA, since the intron-containing genomic DNA sequence would not be amplified.
Note: If primers cannot be designed to separate exons or exon-exon boundaries, it is necessary to treat the RNA sample with RNase-free DNase I or dsDNase in order to remove contaminating genomic DNA.
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Figure 4. Primer design for the qPCR step of RT-qPCR. (1) If one primer is designed to span an exon-intron boundary, the possible contaminating genomic DNA is not amplified, because the primer cannot anneal to the template. In contrast, cDNA does not contain any introns, and is efficiently primed and amplified. (2) When primers flank a long (e.g., 1 kb) intron, the amplification cannot occur because the short extension time is sufficient for the short cDNA sequence but not for the longer genomic target.
A minus reverse transcriptase control (“no RT” control) should be included in all RT-qPCR experiments to test for contaminating DNA (such as genomic DNA or PCR product from a previous run). Such a control contains all the reaction components except for the reverse transcriptase. Reverse transcription should not occur in this control, so if PCR amplification is seen, it is most likely derived from contaminating DNA.
ReferencesQuantitative PCR (qPCR) is a powerful and common technique for accurate analysis of gene expression. Our probe-based qPCR kits are optimized for accurate target quantification and detection over a broad dynamic range, enabling highly reproducible qPCR. These premixes are compatible with all fluorescent oligonucleotide probes or 5' nuclease assays. When deciding whether to do probe-based qPCR or intercalating dye-based qPCR, it is important to consider the advantages and limitations of each method.
Often, probe-based qPCR is used for clinical and diagnostic applications, as it is well-suited for any application that requires maximum specificity and consistent performance. Probe-based qPCR can attain this level of performance because the probes are highly specific for the target sequences and the formation of primer dimers is very rare. Since each probe is uniquely labeled, multiple targets can be amplified in the same tube via multiplexing. This increases throughput, minimizes sample handling, and potentially decreases reagent and consumable usage. Finally, because of the sensitivity and capability of multiplexing, probe-based qPCR works well for genotyping and CNV analyses because the probes can accurately discriminate between different single nucleotide polymorphisms (SNPs) or CNVs.
However, probe-based qPCR has some limitations. While the probes are highly specific, each individual probe can require multiple iterations of design and optimization steps to ensure maximum specificity and performance. Additionally, as the probe is not just an oligonucleotide but contains a fluorophore and a quencher, the probes themselves are often more expensive than simple oligonucleotide primers. While predesigned probes are available, these often have even higher costs.
We offer multiple formats of probe-based qPCR mixes to meet your experimental needs and give you the flexibility to perform a wide variety of applications.
Here are a few examples of research that's been driven by our probe-based qPCR kits:
Jiang, Y. et al. Aberrant expression of RSK4 in breast cancer and its role in the regulation of tumorigenicity. Int. J. Mol. Med. 40, 883–890 ().
Cat. # RR390A was used to accurately and specifically detect altered expression of RSK4 in multiple cell and tissue samples.
Jordan, J. A. & Durso, M. B. Real-time polymerase chain reaction for detecting bacterial DNA directly from blood of neonates being evaluated for sepsis. J. Mol. Diagn. 7, 575–81 ().
Cat. # RR390A was used to develop a rapid assay capable of detecting bacterial DNA directly from blood samples.
Kim, H.-R. et al. Multiplex real-time polymerase chain reaction for the differential detection of porcine circovirus 2 and 3. J. Virol. Methods 250, 11–16 ().
Cat. # RR390A was used to develop a multiplex qPCR assay for the rapid and differential detection of porcine circovirus 2 and 3. This sensitive and reproducible assay had a limit of detection below 50 copies and achieved coefficients of intra-assay and inter-assay variation of less than 4%.
Teng, Q. et al. Development of a TaqMan MGB RT-PCR for the rapid detection of H3 subtype avian influenza virus circulating in China. J. Virol. Methods 217, 64–69 ().
Cat. # RR390A was used with minor groove binder probes to develop a sensitive and rapid assay for H3 avian influenza virus. This assay was 1,000X more sensitive than conventional qPCR, with a detection limit of 10 copies per reaction.