Selecting the appropriate reagents and reaction format is important for achieving specificity and sensitivity in real-time quantitative PCR (qPCR). A general introduction to reverse transcription (RT) reagents, primers, and real-time qPCR reagents such as DNA binding dyes, TaqMan probes, DNA polymerases, and ROX reference dye is presented below.
Related Topics: qPCR Instrumentation, Assay Design and Optimization, MIQE and RDML Guidelines, Real-Time PCR Data Analysis, qPCR Troubleshooting, High Resolution Melting, and PCR Primer and Probe Chemistries.
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Reverse transcription can be combined with qPCR to quantify RNA expression for a sample of interest. Reverse transcriptases are RNA-dependent DNA polymerases that transcribe single-stranded RNA, such as messenger RNA (mRNA) or microRNA (miRNA), into corresponding cDNA. The resulting cDNA is then used as the template for real-time qPCR. This technique is known as RT-qPCR.
One-Step vs. Two-Step RT-qPCR One-step and two-step reaction formats for RT-qPCR are available and refer to whether the RT and real-time qPCR amplification are performed in the same or separate tubes. In the two-step method, RNA is first transcribed into cDNA in a reaction using reverse transcriptase. An aliquot of the resulting cDNA can then be used as a template for multiple qPCR reactions. In the one-step method, RT and qPCR are performed in the same tube.
In two-step RT-qPCR, the RT step can be primed with oligo(dT) primers, random primers, a mixture of the two, or gene-specific primers. One-step RT-qPCR must be performed using gene-specific primers, and can be achieved either by using Thermus thermophilus (Tth) polymerase, a DNA polymerase with inherent RT activity, or by a two-enzyme system combining a reverse transcriptase with a thermostable DNA polymerase. Since Tth DNA polymerase is derived from a thermophilic bacterium, higher temperatures (>60°C) can be used for the RT step, which can minimize secondary structure in high GC-content mRNAs.
Reverse TranscriptasesAccurate analysis of gene expression requires high reproducibility of RT reactions. The robustness of an RT is determined by the sensitivity, dynamic range, and specificity of the reverse transcriptase. Reverse transcriptases from Moloney murine leukemia virus (MMLV) and avian myeloblastoma virus (AMV) are the most commonly used enzymes. When long or full-length cDNA transcripts are needed, MMLV reverse transcriptase and its derivatives are better choices than AMV reverse transcriptase because of their lower RNase H activity.
Primers for Reverse Transcription As noted above, the RT step in two-step RT-qPCR can be performed using oligo(dT) primers, random primers, a mixture of the two, or gene-specific primers. The choice of primers may influence quantification of the target gene. Gene-specific primers produce less background priming than oligo(dT) or random primers. If you choose oligo(dT) primers for RT, you may want to place PCR primers close to the 3' end of the transcript to avoid loss of sensitivity due to truncated messages; this is especially important for longer transcripts. Oligo(dT) priming should be avoided if you are working with transcripts or species that have short poly(A) tails or lack them altogether.
Selecting the appropriate real-time qPCR reagents for an assay depends on several factors including the chemistry used for amplification detection and the real-time instrument platform used.
Real-Time PCR Chemistries
A key step in designing a qPCR assay is selecting the chemistry to monitor the accumulation of amplified target sequence. Fluorescence detection chemistries for qPCR can be categorized into two major types: DNA-binding dyes such as SYBR® Green I, and dye-labeled, sequence-specific oligonucleotide probes such as TaqMan probes.
The chemistry you select for your qPCR assay depends on your application, whether you're performing singleplex or multiplex reactions, and cost considerations. In general, for low-throughput, singleplex experiments, DNA-binding dyes may be preferable because these assays are easier to design and are faster to set up. For high-throughput experiments, however, a fluorescent primer- or probe-based assay (singleplex or multiplex) may be more desirable because the initial cost can be spread over many experiments and the multiplex capability can reduce assay time. Multiplex assays require the use of a fluorescent primer- or probe-based chemistry because the lack of specificity of DNA-binding dyes makes them incompatible with quantitative multiplex assays. Real-time PCR chemistries are covered in more detail in Assay Design and Optimization.
Multiplex reactions require the use of multiple reporters to follow each individual amplification reaction. To distinguish each reaction, choose reporter fluorophores with minimally overlapping emission spectra. The selected fluorophores also need to be compatible with the excitation and emission filters of the real-time instrument.
DNA-Binding Dyes SYBR® Green I and EvaGreen assays use a pair of PCR primers that amplifies a specific region within the target sequence of interest and include a double-stranded DNA (dsDNA)-binding dye for detecting the amplified product. A SYBR® Green I qPCR reaction contains the following components:
Preformulated real-time PCR master mixes containing buffer, DNA polymerase, dNTPs, and dsDNA-binding dye are available from several vendors. Optimized SYBR® Green I qPCR reactions should be sensitive and specific, and should exhibit good amplification efficiency over a broad dynamic range. Since SYBR® Green I binds to all dsDNA, it is necessary to check the specificity of your qPCR assay by analyzing the reaction product(s). To do this, confirm the presence of a single product peak by using the melt curve function on your real-time instrument. You can also run the reaction product on an agarose gel to confirm there is a single DNA band corresponding to the product size of interest.
Real-Time PCR Reagent Selection Guide
• Recommended for use as is – ROX reference setting must be turned "off" + BSA must be added according to instrument specifications
TaqMan Probes A TaqMan probe assay uses a pair of PCR primers and a dual-labeled target-specific fluorescent probe. A TaqMan probe-based qPCR reaction contains the following components:
Preformulated PCR master mixes containing buffer, DNA polymerase, and dNTPs are commercially available from several vendors and can be used in combination with TaqMan probes. As with SYBR® Green I assays, an optimized TaqMan assay should be sensitive and specific, and should exhibit good amplification efficiency over a broad dynamic range.
Primers for Real-Time PCR A successful real-time PCR reaction requires efficient and specific amplification of the DNA product. Both primers and target sequence can affect this efficiency. Therefore, care must be taken when choosing a target sequence and designing primers. A number of free and commercially available software programs exist for this purpose. Primer design programs consider DNA characteristics, such as GC content and secondary structure, to provide the optimal primer sequence for your template. One popular web-based program for primer design is Primer3. Commercially available programs, such as Beacon Designer software, perform both primer design and amplicon selection. Alternatively, predesigned primers are offered from a variety of commercial vendors.
DNA Polymerases During the elongation phase of real-time qPCR, a thermostable DNA polymerase, such as Taq, is used to synthesize the new strand of DNA that is complementary to the template DNA strand. Taq is a common thermostable DNA polymerase, native to the thermophillic bacterium Thermus aquaticus, that has an optimum temperature for activity between ~70–80°C. In addition to Taq, protein engineering has provided a variety of DNA polymerases with features tailored to specific applications such as high-fidelity replication or fast PCR. For example, Sso7d fusion technology has created a highly processive DNA polymerase by covalently linking a nonspecific DNA binding protein to the polymerization domain, thus tethering the polymerase to the DNA during replication. Additionally, both antibody- and chemical-mediated hot-start DNA polymerases help prevent indiscriminate amplification before the reaction cycle begins. Real-time qPCR supermixes, or master mixes, that contain DNA polymerases are available from a variety of vendors.
ROX Reference Dye Certain real-time PCR instrument platforms require an internal reference dye for fluorescent signal normalization and correction of well-to-well optical variations. Variation in fluorescence can result from pipetting error or from instrument design. ROX reference dye is a reagent additive that provides a constant fluorescent signal throughout the real-time qPCR assay for sample normalization.
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