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Technique for stran...
Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription
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Feng, L (författare)
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Lintula, S (författare)
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Ho, TH (författare)
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Anastasina, M (författare)
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Paju, A (författare)
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Haglund, C (författare)
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Stenman, UH (författare)
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Hotakainen, K (författare)
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Orpana, A (författare)
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Kainov, D (författare)
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- Stenman, J (författare)
- Karolinska Institutet
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(creator_code:org_t)
- Future Science Ltd, 2012
- 2012
- Engelska.
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Ingår i: BioTechniques. - : Future Science Ltd. - 1940-9818 .- 0736-6205. ; 52:4, s. 263-
- Relaterad länk:
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https://doi.org/10.2...
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http://kipublication...
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https://doi.org/10.2...
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Abstract
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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°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.
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- Av författaren/redakt...
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Feng, L
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Lintula, S
-
Ho, TH
-
Anastasina, M
-
Paju, A
-
Haglund, C
-
visa fler...
-
Stenman, UH
-
Hotakainen, K
-
Orpana, A
-
Kainov, D
-
Stenman, J
-
visa färre...
- Artiklar i publikationen
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BioTechniques
- Av lärosätet
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Karolinska Institutet