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The activity of influenza RdRp can be measured on several levels. The amplification of each of the viral RNAs during infection can be measured using primer extension or quantitative real-time PCR (qRT-PCR) with primers specific for the segment and sense of the (viral) RNA under investigation. In a more detailed biochemical approach, in vitro assays are available that measure the individual steps carried out by purified RdRp preparations on different templates and using capped or uncapped RNA primers. The main protocols used in the lab are described below. Please contact us with any questions or comments.
This method utilizes reverse transcriptase in conjunction with specific radioactively or fluorescently labelled primers to generate cDNAs from influenza A virus vRNAs, cRNAs, and mRNAs. The difference in sequence and length of the three cDNA products can then be used to separate them on a gel and quantify their relative steady-state levels. The typical protocol can be downloaded here.
In vitro assays can give more detailed information about the enzyme. To purify influenza A virus RNA polymerase from HEK 293T cells use this protocol.
capped oligonucleotide transcription
The activity of purified RdRp preparations can be measured on different templates and different reaction conditions using capped or uncapped RNA primers. This protocol describes the capped primer transcription assay.