Parameters for Successful Reverse Transcription
There are a number of parameters to consider when choosing an RNA extraction method. Although yield is often the primary consideration, genomic DNA (gDNA) removal and inhibitor carryover are also important. When you consider that qPCR detects molecules across a 106 fold range, a purification method with a twofold increase in yield but a lower purity doesn’t offer a real advantage. In fact, choosing a method that gives a higher yield but lower purity, especially with regard to gDNA carryover, can be a poor choice when the sample is intended for RT-qPCR. Even low amounts of gDNA contamination can cause variability in qPCR results, while high amounts of gDNA contamination can cause outright quantitation errors.
One way to help eliminate concerns about gDNA contamination is to include a DNase step in your RNA isolation method. A DNase treatment step can be added at the end of any method. In addition, a no-reverse transcriptase control should always be included in your experiment. Any amplification products from this control are due to the presence of gDNA, and you should assume that the same amount of gDNA is also present in the experimental samples.
Evaluating RNA Quality
Successful reverse transcription depends on RNA integrity and purity. There are several methods for evaluating your RNA before proceeding to the RT step. RNA integrity can be assessed qualitatively by gel electrophoresis or quantitatively using systems such as the Agilent Bioanalyzer, which uses microfluidics to size‐separate and quantitate RNA. If you are concerned about RNA degradation, be sure to follow procedures for creating and maintaining a ribonuclease‐free (RNase‐free) environment to minimize RNA degradation. A good description of these can be found in Blumberg, 1987 (1) . Using an RNase inhibitor (e.g., Recombinant RNasin® Ribonuclease Inhibitor) is strongly recommended.
RNA quantity and purity can be assessed by absorbance. Absorbance at A260 is used to determine RNA amount, and the ratio of absorbance at A260/A280 is used as a measure of purity. High‐quality RNA should have a minimum A260/280 ratio of about 2. Spiking experiments, in which RNA of unknown quality is added to an RT-qPCR that has amplified well in the past, can be used to detect inhibitors. It is a good idea to include a no-RT qPCR control reaction to detect gDNA contamination. Finally, quantifying the RNA will help normalize the amount of RNA you are including in each reaction.
There are three types of RT primers. The first primer anneals specifically to the poly(A) portion of the 3´-end of the transcript (oligo(dT) primer). Oligo(dT) primers can be problematic when your target region is at the 5´-ends of long mRNAs or when the molecules have significant secondary structure, which can cause the reverse transcriptase to stall during cDNA synthesis. The second type of primer is random hexamer primers. These can prime reverse transcription at multiple points along the transcript, and for this reason, they are useful for long mRNAs and transcripts with significant secondary structure. Gene-specific primers often are used in one-step (coupled) RT-PCR.
For RT-qPCR applications where the final amplicon length is about 100bp or less, using higher concentrations of random primers may be advantageous. You will have a higher probability of a priming event at the 3´ end of the mRNA, a greater chance of multiple cDNAs produced from each transcript and a higher probability of priming rare transcripts. Regardless of primer choice, the final primer concentration may need to be optimized.
One-Step versus Two-Step RT-PCR
RT-PCR can be performed using one of two strategies: one-step RT-PCR or two-step RT-PCR. The fundamental difference between these two approaches is whether the RT step is performed separately from the PCR or in the same tube. For one-step RT-PCR, the RT and PCR steps are performed sequentially in the same tube using the entire amount of the cDNA synthesis products as the template for PCR. For two-step RT-PCR, the RT and PCR steps are also performed sequentially, but only a portion of the cDNA products is used as a template for PCR, which is performed in a separate tube. For this reason, one-step RT-PCR may be the more sensitive approach. However, two-step RT-PCR allows multiple PCRs from a single RT reaction, which works well for quantifying multiple targets or for doing replicate assays.
One-step RT-PCR commonly uses gene-specific primers for both the RT and PCR steps, with one of the PCR primers also acting as the RT primer. Two-step RT-qPCR can use oligo(dT) primers or random primers for the RT step, but a sequence-specific primer also can be used.
Real-Time PCR Approaches
Fluorescent DNA-Binding Dyes
Using fluorescent DNA-binding dyes is one of the easiest qPCR approaches to detect amplicons in real time. The dye is simply added to the reaction, and fluorescence is measured at each PCR cycle. Because the fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored as an increase in fluorescent signal. However, preliminary work often must be done to ensure that the PCR conditions yield only specific product. In subsequent reactions, specific amplification can be verified by a melt curve analysis. Thermal melt curves are generated by allowing all product to form double-stranded DNA at a lower temperature (approximately 60°C) and slowly ramping the temperature to denaturing levels (approximately 95°C). The product length and sequence affect melting temperature (Tm), so the melt curve is used to characterize amplicon homogeneity. Nonspecific amplification can be identified by broad peaks in the melt curve or peaks with unexpected Tm values. By distinguishing specific and nonspecific amplification products, the melt curve adds a quality control aspect during routine use. However, it is not possible to generate melting curves with assays that rely on the 5´→3´ exonuclease activity of Taq DNA polymerase, such as the probe-based TaqMan® technology.
An example of a qPCR technology that uses a DNA-binding dye is the GoTaq® qPCR Master Mix (Cat.# A6001). This system contains a proprietary fluorescent DNA-binding dye that often exhibits greater fluorescence enhancement upon binding to double-stranded DNA and less PCR inhibition than the commonly used SYBR® Green I dye, while using the same filters and settings for detection. The dye is included in the GoTaq® qPCR Master Mix and does not inhibit amplification, resulting in earlier quantification cycle values and an expanded linear range (Figure 1). For more information, view the GoTaq® qPCR Master Mix video.
Labeled Primers or Probes
Real-time PCR using labeled oligonucleotide primers or probes employs two different fluorescent reporters and relies on energy transfer from one reporter (the energy donor) to a second reporter (the energy acceptor) when the reporters are in close proximity. The second reporter can be a quencher or a fluor. If the second reporter is a quencher, the energy from the first reporter is absorbed but re-emitted as heat rather than light, leading to a decrease in fluorescent signal. Alternatively, if the second reporter is a fluor, the energy can be absorbed and re-emitted at another wavelength through fluorescent resonance energy transfer (FRET, reviewed in Didenko, 2001 (2) ). During the exponential phase of PCR, the change in fluorescence is proportional to accumulation of PCR product. Different assay designs utilize quenching/dequenching or FRET in different ways to follow product accumulation. Some examples are presented below.
One example of a primer-based approach is the Plexor® qRT-PCR Systems, which require two PCR primers, only one of which is fluorescently labeled. These systems take advantage of the specific interaction between two modified nucleotides (3) (4) (5) . The two novel bases, isoguanine (iso-dG) and 5´-methylisocytosine (iso-dC), form a unique base pair in double-stranded DNA (4) . To perform fluorescent quantitative PCR using this new technology, one primer is synthesized with an iso-dC residue as the 5´-terminal nucleotide and a fluorescent label at the 5´-end; the second primer is unlabeled. During PCR, this labeled primer is annealed and extended, becoming part of the template used during subsequent rounds of amplification. The complementary iso-dGTP, which is available in the nucleotide mix as dabcyl-iso-dGTP, pairs specifically with iso-dC. When the dabcyl-iso-dGTP is incorporated, the close proximity of the dabcyl quencher and the fluorescent label on the opposite strand effectively quenches the fluorescent signal. This process is illustrated in Figure 2. The initial fluorescence level of the labeled primers is high in Plexor® System reactions. As amplification product accumulates, signal decreases.
Probe-Based qPCR Methods
Some qPCR strategies employ complementary nucleic acid probes to quantify the DNA target. These probes also can be used to detect single nucleotide polymorphisms (6) (7) . There are several general categories of real-time PCR probes, including hydrolysis, hairpin and simple hybridization probes. These probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but probes can differ in the number and location of the fluorescent reporters. The use of simple hybridization probes involves two labeled probes or, alternatively, one labeled probe and a labeled PCR primer. In the first approach, the energy emitted by the fluor on one probe is absorbed by a fluor on the second probe, which hybridizes nearby. In the second approach, the emitted energy is absorbed by a second fluor that is incorporated into the PCR product as part of the primer. Both of these approaches result in increased fluorescence of the energy acceptor and decreased fluorescence of the energy donor.
Label-Based qPCR Methods
A benefit of the label-based methods over those using DNA-binding dyes is the capacity for multiplexing. While DNA-binding dyes are not sequence-specific, label‐based approaches are sequence‐specific because the fluorescent marker is on the amplification primer and only produces a strong signal when bound to the DNA target. The labeled primer can be tagged with one of many common fluorescent labels, allowing two- to four-color multiplexing, depending on the instrument used. This means that you can analyze both your target and control gene in the same well. Finally, the simplicity of primer design for the labeled primer-based method is a distinct advantage over probe-based quantitative PCR approaches.