title: Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription.
abstract: 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.
authors.affiliation: Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Lin
authors.initials: L
authors.lastname: Feng
authors.forename: Susanna
authors.initials: S
authors.lastname: Lintula
authors.forename: Tho Huu
authors.initials: TH
authors.lastname: Ho
authors.forename: Maria
authors.initials: M
authors.lastname: Anastasina
authors.forename: Annukka
authors.initials: A
authors.lastname: Paju
authors.forename: Caj
authors.initials: C
authors.lastname: Haglund
authors.forename: Ulf-Håkan
authors.initials: UH
authors.lastname: Stenman
authors.forename: Kristina
authors.initials: K
authors.lastname: Hotakainen
authors.forename: Arto
authors.initials: A
authors.lastname: Orpana
authors.forename: Denis
authors.initials: D
authors.lastname: Kainov
authors.forename: Jakob
authors.initials: J
authors.lastname: Stenman
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis*; D004285:Dogs; D005786:Gene Expression Regulation; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription / Q000235:genetics*; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; C057027:PB2 protein, Influenzavirus A; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase