To demonstrate the utility of this technique, we applied it for analysis of viral PB2 negative sense RNA (-, or vRNA) and positive-sense RNA (+, cRNA and mRNA) during virus replication. For this purpose MDCK cells were infected with influenza A virus at MOI 3 and harvested immediately or 3, 6, 9, and 12 h postinfection (hpi). Total RNA was purified from the infected cells. For unambiguous detection of (+) and (-) RNA strands, a pair of strand-specific sequence-modifying reverse transcription primers were designed in such a manner that generated cDNA would be amplified by a common pair of PCR primers, but the melting temperatures of the (+) and (-) PCR amplicons would differ by 8°C (Supplementary Figure S2). Ten thousand copies of plasmid carrying the unmodified PB2 sequence were added to each PCR as an internal standard. Thus, three separate peaks were generated during postamplification melting curve analysis representing PCR amplicons derived from (+) RNA, plasmid DNA and (-) RNA, respectively (Figure 4). At the time of infection (0 hpi) there was no amplification derived from viral RNA, and only the peak from the 10,000 copies of internal standard could be detected. Expression of PB2 mRNA increased sharply at 3 hpi (33,649 copies) causing competitive depression of the internal standard. The amount of PB2 vRNA gradually increased from 6 hpi (305,476 copies) to dominate the 9 hpi and 12 hpi amplification reactions. Since all three PCR products share the same PCR primers, the amplification efficiency of these amplicons are close to identical and quantitative analysis can be done accurately by comparing the area of the respective melting curve peaks. The linear range of the PB2 standard curve PCR was 10–108 copies. However, there was a 2°C shift of the melting temperature at 10 copies, and we consider 100 copies of cDNA is the limit for reliable quantification of cDNA. We have previously shown that the sensitivity of this type of RT-PCR assays is similar to TaqMan-based qRT-PCR. The dynamic range for relative quantification of two competitively amplified amplicons was about three orders of magnitude (20). By allowing simultaneous quantification of the different strands of viral RNA this technique provides a useful tool for studying the dynamics of viral RNA transcription and replication. The finding that PB2 (+) RNA expression occurs early after infection, while the amount (-) RNA as a result of replication increases more slowly to dominate the amplification reactions from later time points post infection is in concordance with earlier reports (22)(23). A limitation of this technique is that it cannot, as such, distinguish mRNA from cRNA.
Performing reverse transcription at high temperatures has been shown to improve the strand-specificity of RT-PCR, by reducing nonspecific priming in the reverse transcription step (21)(24). These findings indicate that even when using specific primers for initiating cDNA synthesis, a significant part of the generated cDNA might indeed be nonspecifically primed by primers binding to false priming sites or by short nucleic acid sequences and in particular by 3′ secondary structures present in the mRNA itself (4)(21). Although the thermal stability of RNA secondary structures can vary considerably depending on sequence length and base constitution, the maximal binding energy of small oligonucleotide molecules and free 3′-end capable of priming reverse transcription is theoretically limited. For this reason, increasing reverse transcription temperature appears as a potential means for controlling nonspecific priming. Thermo-stable Tth reverse transcriptase has been used to perform reverse transcription at temperatures up to 70°C to improve strand-specific detection of viral RNA (25)(26). Another study showed significant improvement of strand specificity of the RT reaction, using an RNase H+ reverse transcription enzyme and performing cDNA synthesis at 50°C (21). Regardless of which approach is chosen, there seems to be considerable differences in the rate of nonspecific priming between different RNA templates.
The technique described here provides a quantitative tool for strand-specific mRNA expression analysis and allows for monitoring of the proportion of primer-independent cDNA synthesis in every separate reverse transcription reaction. We have shown that while there are pronounced differences in tendency toward nonspecific priming between different target sequences, the overall specificity of reverse transcription can be increased by raising the reaction temperature to 55°–60°C, without significant loss of sensitivity. We have demonstrated the utility of this technique for strand-specific analysis of viral RNA. Analogous design of sequence-modifying primers enables simultaneous analysis of bidirectional RNA synthesis in eukaryotic cells.
We thank Anne Ahmanheimo and Laura Mäkelä for excellent technical support. We would also like to thank Mr. Mikko Turunen for providing the Gp5d and Lovo cell lines. This work was supported by the Finnish Funding Agency for Technology and Innovation (TEKES), the Minerva Institute for Medical Research, the Jane and Aatos Erkko Foundation, and Medicinska Understödsföreningen Liv och Hälsa.
The authors declare no competing interests.
Address correspondence to Jakob Stenman, Institute for Molecular Medicine Finland Fimm, Biomedicum Helsinki 2U room G315b, Tukholmankatu 8, 00290 Helsinki, Finland. Email: [email protected]
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