This section provides an overview of the major reaction components and parameters involved in real-time PCR experiments. A more detailed discussion of specific components like reporter dyes, passive reference dyes, and uracil DNA glycosylase (UDG) is provided in subsequent sections of this handbook.
PCR performance is often related to the thermostable DNA polymerase, so enzyme selection is critical to success. One of the main factors affecting PCR specificity is the fact that Taq DNA polymerase has residual activity at low temperatures.
Primers can anneal nonspecifically to DNA during reaction setup, allowing the polymerase to synthesize nonspecific product. The problem of nonspecific products resulting from mis-priming can be minimized by using a “hot-start” enzyme.
Using a hot-start enzyme ensures that DNA polymerase is not active during reaction setup and the initial DNA denaturation step.
The reverse transcriptase (RT) is as critical to the success of qRT-PCR as the DNA polymerase. It is important to choose an RT that not only provides high yields of full-length cDNA, but also has good activity at high temperatures.
High-temperature performance is also very important for denaturation of RNA with secondary structure. In one-step qRT-PCR, an RT that retains its activity at higher temperatures allows you to use a GSP with a high melting temperature (Tm), increasing specificity and reducing background.
It is a good idea to purchase both the dNTPs and the thermostable DNA polymerase from the same vendor, as it is not uncommon to see a loss in sensitivity of one full threshold cycle (Ct) in experiments that employ these reagents from separate vendors.
In real-time PCR, magnesium chloride or magnesium sulfate is typically used at a final concentration of 3 mM.
This concentration works well for most targets; however, the optimal magnesium concentration may vary between 3 and 6 mM.
Good experimental technique
Do not underestimate the importance of good laboratory technique. It is best to use dedicated equipment and solutions for each stage of the reactions, from preparation of the template to post-PCR analysis.
The use of aerosol-barrier tips and screwcap tubes can help decrease cross-contamination problems. To obtain tight data from replicates (ideally, triplicates), prepare a master mix that contains all the reaction components except sample.
The use of a master mix reduces the number of pipetting steps and, consequently, reduces the chances of cross-well contamination and other pipetting errors.
Use 10 to 1,000 copies of template nucleic acid for each real-time PCR reaction. This is equivalent to approximately 100 pg to 1 μg of genomic DNA, or cDNA generated from 1 pg to 100 ng of total RNA.
Excess template may also bring higher contaminant levels that can greatly reduce PCR efficiency.
Depending on the specificity of the PCR primers for cDNA rather than genomic DNA, it may be important to treat RNA templates to reduce the chance that they contain genomic DNA contamination. One option is to treat the template with DNase I.
Pure, intact RNA is essential for full-length, high-quality cDNA synthesis and may be important for accurate mRNA quantification.
RNA should be devoid of any RNase contamination, and aseptic conditions should be maintained. Total RNA typically works well in qRT-PCR; isolation of mRNA is typically not necessary, although it may improve the yield of specific cDNAs.
Real-time PCR primer design
Good primer design is one of the most important parameters in real-time PCR. This is why many researchers choose to purchase TaqMan® Assay products-primers and probes for real-time PCR designed using a proven algorithm and trusted by scientists around the world.
If you choose to design your own real-time PCR primers, keep in mind that the amplicon length should be approximately 50–150 bp, since longer products do not amplify as efficiently.
In general, primers should be 18–24 nucleotides in length. This provides for practical annealing temperatures.
Primers should be designed according to standard PCR guidelines. They should be specific for the target sequence and be free of internal secondary structure. Primers should avoid stretches of homopolymer sequences (e.g., poly(dG)) or repeating motifs, as these can hybridize inappropriately.
Primer pairs should have compatible melting temperatures (within 1°C) and contain approximately 50% GC content. Primers with high GC content can form stable imperfect hybrids.
Conversely, high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3’ end of the primer should be GC rich to enhance annealing of the end that will be extended. Analyze primer pair sequences to avoid complementarity and hybridization between primers (primer-dimers).
For qRT-PCR, design primers that anneal to exons on both sides of an intron (or span an exon/exon boundary of the mRNA) to allow differentiation between amplification of cDNA and potential contaminating genomic DNA by melting curve analysis.
To confirm the specificity of your primers, perform a BLAST® search against public databases to be sure that your primers only recognize the target of interest.
Optimal results may require a titration of primer concentrations between 50 and 500 nM. A final concentration of 200 nM for each primer is effective for most reactions.
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