Real time RT PCR

Guidelines for RT-PCR

When performing real-time RT-PCR, the primers and the enzyme for reverse transcription must be carefully chosen. The primers should allow reverse transcription of all targets of interest, and the reverse transcriptase should yield cDNA amounts that accurately represent the original RNA amounts to ensure accurate quantification. In addition, the effects of the components of the RT reaction on subsequent real-time PCR must be minimized.

To perform PCR using RNA as a starting template, the RNA must first be reverse transcribed into cDNA in a reverse transcription (RT) reaction. RT and PCR can be carried out either sequentially in the same tube (one-step RT-PCR) or separately (two-step RT-PCR). One-step RT-PCR requires gene-specific primers.

Two-step and one-step RT-PCR

Real-time RT-PCR can take place in a two-step or one-step reaction (see figure Comparison of two-step and one-step RT-PCR and table Advantages of different RT-PCR procedures). With two-step RT-PCR, the RNA is first reverse transcribed into cDNA using oligo-dT primers, random oligomers, or gene-specific primers. An aliquot of the reverse-transcription reaction is then added to the real-time PCR. It is possible to choose between different types of RT primers, depending on experimental needs. Use of oligo-dT primers or random oligomers for reverse transcription means that several different transcripts can be analyzed by PCR from a single RT reaction. In addition, precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage.

Comparison of two-step and one-step RT-PCR and table Advantages of different RT-PCR procedures

In one-step RT-PCR — also referred to as one-tube RT-PCR — both reverse transcription and real-time PCR take place in the same tube, with reverse transcription preceding PCR. This is possible due to specialized reaction chemistries and cycling protocols (see Conditions for one-step RT-PCR). The fast procedure enables rapid processing of multiple samples and is easy to automate. The reduced number of handling steps results in high reproducibility from sample to sample and minimizes the risk of contamination since less manipulation is required.

Advantages of different RT-PCR procedures

Procedure	Advantages
Two-step RT-PCR	Multiple PCRs from a single RT reaction Flexibility with RT primer choice Enables long-term storage of cDNA
One-step RT-PCR	Easy handling Fast procedure High reproducibility Low contamination risk

Choice of RT primers

The choice of primers for reverse transcription depends on whether one-step or two-step RT-PCR is being carried out. In one-step RT-PCR, the downstream PCR primer is also the primer for reverse transcription. Therefore, one-step RT-PCR is always performed with gene-specific primers. In two-step RT-PCR, 3 types of primers, and mixtures thereof, can be used for reverse transcription: oligo-dT primers (typically 13–18mers), random oligomers (such as hexamers, octamers, or nonamers), or gene-specific primers (see table “Suitability of primer types for RT-PCR”). If oligo-dT primers are used, only mRNAs will be reverse transcribed starting from the poly-A tail at the 3' end. Random oligomers will enable reverse transcription from the entire RNA population, including ribosomal RNA, transfer RNA, and small nuclear RNAs. Since reverse transcription is initiated from several positions within the RNA molecule, this will lead to relatively short cDNA molecules. In comparison, gene-specific primers allow reverse transcription of a specific transcript.

A universal priming method for the RT step of real-time two-step RT-PCR should allow amplification and detection of any PCR product regardless of transcript length and amplicon position, and achieve this with high sensitivity and reproducibility.

Suitability of primer types for RT-PCR

Application	Recommended type of primer
RT-PCR of specific transcript	Gene-specific primer gives highest selectivity and only the RNA molecule of choice will be reverse transcribed
RT-PCR of long amplicon	Oligo-dT or gene-specific primers
RT-PCR of an amplicon within long transcript	Gene-specific primers, random oligomers, or a mixture of oligo-dT primers and random nonamers are recommended so that cDNA covering the complete transcript is produced

Conditions for two-step RT-PCR

Effect of RT volume added to two-step RT-PCR

In two-step RT-PCR, the addition of the completed reverse-transcription reaction to the subsequent amplification reaction transfers not only cDNA template, but also salts, dNTPs, and RT enzyme. The RT reaction buffer, which has a different salt composition to that of the real-time PCR buffer, can adversely affect real-time PCR performance. However, if the RT reaction forms 10% or less of the final real-time PCR volume, performance will not be significantly affected. Use of 3 µl of RT reaction in a 20 µl PCR (i.e., 15% of the final volume) can lead to significant inhibition of real-time PCR. We recommend testing dilutions of the RT reaction in real-time PCR to check the linearity of the assay. This helps to eliminate any inhibitory effects of the RT reaction mix that might affect accurate transcript quantification.

Effect of RNA secondary structure

RNA secondary structure can affect RT-PCR in several ways. Regions of RNA with complex secondary structure can cause the reverse transcriptase to stop or dissociate from the RNA template (see figure Effects of complex secondary structure on RT-PCR: RT effects).

Effects of complex secondary structure on RT-PCR: RT effects

The truncated cDNAs, missing the downstream primer-binding site, are then not amplified during PCR. Alternatively, the reverse transcriptase can skip over looped-out regions of RNA, which are then excluded from the synthesized cDNA. In the PCR step, these cDNAs with internal deletions are amplified and appear as shortened PCR products. Ideally, the reverse transcriptase should not be affected by RNA secondary structure and should be capable of reverse transcribing any template, without the need for reaction optimization.

With high GC content, the tight association of RNA:DNA hybrids can interfere with primer binding during PCR and prevent DNA polymerases from progressing (see figure Effect of high GC content on RT-PCR: PCR effects). RNase H removes RNA in RNA:DNA hybrids to allow primer binding and second-strand DNA synthesis. RNase H digestion has been previously shown to improve RT-PCR yield and to be required for amplification of some sequences, even as short as 157 bp (7).

Conditions for one-step RT-PCR

The ideal reverse transcriptase for one-step RT-PCR should also exhibit the same properties as those described above for reverse transcriptases for two-step RT-PCR. However, one of the main problems in one-step RT-PCR is the inhibitory effect of the reverse transcriptase on the PCR step, which can lead to increased CT values and thus reduced sensitivity and specificity when compared with two-step RT-PCR.

RT-PCR primer design

A critical factor in RT-PCR is the selection of appropriate primers for maximal efficiency and specificity. Primer specificity is affected by a number of factors, including sequence, primer location, and the RT-PCR system used. General primer-design rules for PCR are also applicable in RT-PCR to avoid mispriming and primer–dimer formation (see PCR primer design). These effects are even more pronounced in RT-PCR, where cDNAs produced during reverse transcription are more susceptible to nonspecific priming due to their single-stranded nature. Nonspecific priming in RT-PCR reduces the sensitivity of the process, leading to reduced yields of specific products or failure of the RT-PCR altogether.

To avoid amplification of contaminating genomic DNA, primers for RT-PCR should be designed so that one half of the primer hybridizes to the 3' end of one exon and the other half to the 5' end of the adjacent exon (see figure RT-PCR primer design). Such primers will anneal to cDNA synthesized from spliced mRNAs, but not to genomic DNA.

RT-PCR primer design

To detect amplification of contaminating DNA, RT-PCR primers should be designed to flank a region that contains at least one intron. Products amplified from cDNA (no introns) will be smaller than those amplified from genomic DNA (containing introns). Size difference in products is used to detect the presence of contaminating DNA.

If only the mRNA sequence is known, choose primer annealing sites that are at least 300–400 bp apart. It is likely that fragments of this size from eukaryotic DNA contain splice junctions. As explained in the previous point, such primers may be used to detect DNA contamination.

In summary, the following factors should be considered when designing primers for RT-PCR:

Annealing temperature can affect RT-PCR efficiency and sensitivity.

High primer concentrations can cause mispriming and primer–dimer formation.

A stringent hot start is essential for optimal RT-PCR performance.

Primer design in RT-PCR allows differentiation of signals from RNA and contaminating DNA. For best results, DNA-free RNA should be used in order to avoid competition of DNA in RT-PCR.

Enzymes used in RT-PCR

RT-PCR allows the analysis of RNA using a combination of reverse transcription and PCR. cDNA is synthesized from RNA templates using reverse transcriptases — RNA-dependent DNA polymerases normally isolated from a variety of retroviral sources (e.g., from Avian Myeloblastosis Virus [AMV] or Moloney murine leukemia virus [MMLV]).

Although thermostable DNA polymerases such as Tth DNA polymerase also exhibit reverse transcriptase activity under specific conditions, these enzymes are not as efficient for reverse transcription as mesophilic reverse transcriptases.

The single-stranded cDNA produced by reverse transcription is more susceptible to nonspecific primer annealing at lower temperatures than double-stranded DNA (e.g., genomic DNA). Nonspecific annealing can result in poor amplification specificity which, especially when combined with limiting cDNA quantity or low transcript abundance, leads to reduced sensitivity and poor reproducibility. Amplification specificity is crucial for successful RT-PCR and is best achieved by combining innovative buffer solutions with specially modified reverse transcriptases and hot-start PCR.

Multiplex PCR and RT-PCR

In multiplex, real-time PCR, several genomic DNA targets are quantified simultaneously in the same reaction. Multiplex, real-time RT-PCR is a similar method, allowing simultaneous quantification of several RNA targets in the same reaction. The procedure can be performed either as two-step RT-PCR or as one-step RT-PCR.

Multiplex PCR and RT-PCR offer many advantages for applications such as gene expression analysis, viral load monitoring, and genotyping. The target gene(s) as well as an internal control are co-amplified in the same reaction, eliminating the well-to-well variability that would occur if separate amplification reactions were carried out. The internal control can be either an endogenous gene that does not vary in expression between different samples (e.g., a housekeeping gene; see table Housekeeping genes commonly used as endogenous references) or an exogenous nucleic acid. For viral load monitoring, the use of an exogenous nucleic acid as internal control allows the following parameters to be checked: the success of sample preparation, the absence of inhibitors, and the success of PCR. Multiplex analysis ensures high precision in relative gene quantification, where the amount of a target gene is normalized to the amount of a control reference gene. Quantification of multiple genes in a single reaction also reduces reagent costs, conserves precious sample material, and allows increased throughput.

Multiplex PCR and RT-PCR are made possible by the use of sequence-specific probes that are each labeled with a distinct fluorescent dye and an appropriate quencher moiety. This means that the emission maxima of the dyes must be clearly separated and must not overlap with each other. In addition, reactions must be carried out on an appropriate real-time cycler that supports multiplex analysis (i.e., the excitation and detection of several non-overlapping dyes in the same well or tube).