Reverse transcription at 42°C was performed with the M-MulV RNase H- reverse transcriptase (Fisher Scientific Finnzymes, Espoo, Finland). At higher reaction temperatures, RevertAid Premium Reverse Transcriptase (Thermo Scientific Fermentas, St. Leon-Ro,Germany) was used according to the manufacturer's instructions. An equal amount of total RNA from the three cultured cells was mixed and used as a template in the reverse transcription reactions. For M-MuLV RT, cDNA synthesis was carried out in a 20-µL reaction volume containing 10 µL χ 2× reverse transcription buffer, 10 pmol × each of the sequence-modifying antisense primers (see Supplementary Table S1), 800 ng of the RNA mixture, and 2 µL M-MuLV RNase H- reverse transcriptase. The reverse transcription reaction was incubated at 42°C for 50 min, and then the reverse transcriptase was inactivated for 5 min at 85°C. For RevertAid Premium RT, 800 ng RNA mixture were transcribed in 20-µL reaction volume containing 4 µL of 5× reverse transcription buffer, 1 µL dNTP mix (0.5 mM final concentration), 10 pmol each of the sequence-modifying antisense primers, 0.5 µL of RNase inhibitor (Thermo Scientific Fermentas) and 1 µL RevertAid Premium Reverse Transcriptase. The reverse transcription reaction was incubated first at 75°C for 5 min to open RNA secondary structures. The samples were cooled to reverse transcription temperature, and RevertAid Premium RT was added to the reactions. Reverse transcription was carried out at 50°, 55°, or 60° for 30 min, and the reverse transcriptase was inactivated at 85° for 5 min. Reverse transcriptase-negative and RNA template-negative controls were carried out in parallel for each of the analyzed samples. Sequence-modifying reverse transcription of the H1N1 PB2 gene was performed with 100 ng purified total RNA, at 55°C for 30 min.Exonuclease I treatment
After reverse transcription, the unbound sequence-modifying reverse transcription primers were inactivated by exonuclease treatment. 40 units of Exonuclease I (New England Biolabs GmbH, Frankfurt-Hoechst, Germany) was added to the reverse transcription reactions. Exonuclease digestion was performed at 42°C for 45 min, followed by denaturation at 80°C for 15 min.PCR and melting curve analysis
One µL Exonuclease I-treated reverse transcription products was amplified with the DyNAmo HS SYBR Green PCR kit (Fisher Scientific Finnzymes) according to the manufacturer's instructions. qPCR amplification was performed on the ABI 7500 Fast instrument (Applied Biosystems, Carlsbad, CA, USA). cDNA products were amplified for 35 cycles, with 15 s denaturation at 95°C and 1 min annealing at 57°C. For viral RNA samples, an additional 10,000 copies of control plasmids were added to each PCR. Melting curve acquisition and analysis was carried out immediately after amplification, by an additional denaturation at 95°C and continuous melting curve acquisition from 57° to 95°C with a 0.1°C/s ramp rate and measuring points at 0.3°C intervals. A first derivative melting curve plot was obtained by use of the default settings of the instrument.Results and discussion
Previous studies have shown that primer-independent cDNA synthesis occurs commonly in RT-PCR assays and can contribute to a significant part of the final amplification product (4-6)(17-19)(21). To determine the rate of primer-independent cDNA synthesis at standard reverse transcription conditions, we carried out reverse transcription without addition of exogenous primers using M-MuLV RNase H- reverse transcriptase at 42°C. Total RNA from a mixture of three human colon adenocarcinoma cell lines was used as template, and reverse transcriptase negative controls were included for each of the samples. The cDNA synthesis products were amplified by qPCR using PCR primers for 13 potential colorectal cancer marker genes and two housekeeping genes. PCR product specificity was verified by postamplification melting curve analysis. Purified human genomic DNA (gDNA) was included as a universal positive control for each of the analyzed genes in the PCR step. We observed a consistent PCR amplification product from the all the 15tested genes, with Ct values in the range of 16 to 31. The melting temperature of the PCR products corresponded exactly to that of the PCR products from the gDNA controls. Contamination by genomic DNA or previous amplification products was excluded, as no amplification was observed in any of the reactions originating from reverse transcriptase negative controls. These results indicate that the observed PCR product was reverse transcriptase-dependent and that the template for this amplification was nonspecifically primed cDNA.