2Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland
3Institute for Molecular Medicine Finland FIMM, Helsinki, Finland
4Helsinki University Central Hospital, HUSLAB, Helsinki, Finland
5Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland
6Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%–57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.
Reverse-transcription PCR (RT-PCR) remains the most sensitive technique for low copy number nucleic acid detection (1). While there are several ways of controlling amplification reaction efficiency and specificity, a large proportion of the variation observed in RT-PCR assays has been attributed to the reverse transcription step (2). Specific priming and multiplex specific priming have been compared with random hexamers as well as oligo(dt) primers, but superiority of one approach over another seems to be template-dependent (2)(3). Primer-independent reverse transcription has been reported to complicate strand-specific detection of viral mRNA as cDNA derived from the undesired viral RNA (vRNA) strand is cogenerated during reverse transcription and is subsequently amplified in the PCR step (4-6). The same phenomenon impedes strand-specific expression analysis in eukaryotic cells (7-12). There is increasing evidence suggesting that bidirectional transcription composes a substantial part of the eukaryotic transcriptome (7)(13-16). Antisense transcription functions actively in gene silencing, degradation of the corresponding sense transcripts, as well as in expression of coding-RNA (7). The lack of strand specificity during reverse transcription has been shown to be caused by primer-independent priming of the incorrect strand and is thought to be induced by various reasons, such as RNA secondary hairpin structures or random-priming by either cellular or exogenous small nucleic acids (17-19).
We have developed an RT-PCR based assay, for intron-independent quantitative gene expression analysis (20). The kernel of this technology is a unior multiplex reverse-transcription reaction with sequence-modifying primers. These primers alter the sequence of specifically primed cDNA transcripts by substituting nucleotides adenine or thymine to cytosine or guanine, or vice versa. The modification gives rise to a 3°–5°C change in melting temperature of the subsequent PCR amplicon. PCR amplification is performed in separate gene-specific reactions using a standard quantitative PCR (qPCR) protocol. Subsequent separation and detection of PCR products derived from specifically primed cDNA and primer-independent cDNA synthesis is carried out by end-point melting curve analysis. Because of the sequence modification of all primer-initiated cDNA transcripts, this technique provides a means to quantitatively monitor the proportion of nonspecific primer-independent cDNA synthesis that occurs during reverse transcription. We have utilized this technique to optimize the specificity of the reverse transcription reaction and applied it for strand-specific analysis of viral RNA in influenza A virus infected cells.Materials and methods Cell culture
Colo-205 cells were initially obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). Lovo and Gp5d cells were obtained from ECACC (European Collection of Cell Cultures, Salisbury, UK). Cells were grown at 37°C in a humidified atmosphere with 5% CO2. Colo-205 cells were maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum, 2 µM L-glutamine, 100,000 units/L penicillin, and 100 mg/L streptomycin. Lovo cells and Gp5d cells were maintained in Ham's F12 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum, 2 µM L-glutamine, 100,000 units/L penicillin, 100 mg/L streptomycin. MDCK cells were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplied with 10% heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin mix (PenStrep mix, 500 U each) (Lonza, Walkersville, MD, USA), and normocin (0.1 mg/mL). Cells were grown in 37°C in 5% CO2.Viruses, cell lines, and viral infections
Human Influenza A virus A/WSN/33 (H1N1) was propagated in MDCK cell line. For infection, cells were washed with phosphate-buffered saline (PBS) twice. Virus at a multiplicity of infection (MOI) 3 was added in DMEM supplied with 10% BSA, PenStrep mix, and TPCK Trypsin (Sigma-Aldrich) at final concentration 1.0 µg/mL. To harvest, cells were treated with 10× trypsin with EDTA (Lonza), pelleted, washed twice with PBS, and were either frozen in -80°C or immediately used in further experiments.RNA extraction
Total RNA was extracted from the cultured cells by RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Following extraction the RNA was DNase I-treated by adding 10 units of DNase I (Roche Diagnostics, Rotkreuz, Switzerland) to 10 µg RNA, 5 µL 10× DNase buffer (100 mmol/L Tris, pH 7.5, 25 µM MgCl2, 5 mmol/L CaCl2), and incubating for 30 min at 37°C. After the incubation the DNase I was inactivated for 5 min at 70°C. RNA was then precipitated with 5 µL 2 µM sodium acetate (pH 4.0) and 140 µL of 100% ethanol overnight at –20°C, after which the sample was centrifuged at 17, 900× g for 30 min at 4°C. The supernatant was removed carefully and the RNA pellet was washed with 200 µL 70% ethanol, after which the sample was centrifuged at 17, 900× g for 20 min at 4°C. The supernatant was removed, and the RNA was dissolved in 20 µL RNase-free water. RNA concentrations and quality were assessed by absorbance (NanoDrop, Wilmington, DE, USA).