Journal
BIOTECHNIQUES
Volume 52, Issue 4, Pages 263-+Publisher
BIOTECHNIQUES OFFICE
DOI: 10.2144/0000113842
Keywords
RT-PCR; primer-independent reverse transcription; proportion
Funding
- Finnish Funding Agency for Technology and Innovation (TEKES)
- Minerva Institute for Medical Research
- Jane and Aatos Erkko Foundation
- Medicinska Understodsforeningen Liv och Halsa
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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 degrees C and accounted for 11%-57% of the final PCR amplification products. By raising the reaction temperature to 55 degrees 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.
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