RNA extraction
Extraction of nucleic acids from swabs using the QIAGEN BioRobot Universal
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This protocol details the requirements for the extraction of RNA from swabs and tissues collected to detect the presence of influenza virus and Newcastle disease virus (NDV). The RNA can be extracted from tissues, cloacal, tracheal or nasal swabs. Wooden or charcoal swabs are not suitable for testing.
Dry swabs are introduced into 1ml of brain heart infusion broth or L-15 medium with antibiotics (BHIB), but virus transport media cannot be used. Extraction of the RNA is carried out in a QIAGEN BioRobot Universal robotic machine, and the RNA product should be clear and colourless.
The extracted RNA can be subjected to Real-Time reverse transcription-PCR (RRT-PCR) to assess presence of influenza A viruses and avian paramyxovirus type 1 (APMV-1) in the original sample.
Manual RNA extraction from biological samples
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This protocol details the requirement for extraction of RNA from biological samples using commercial extraction kits. The RNA extraction kits can be used for the extraction of RNA from a range of samples including
clarified tissue homogenate supernatants
egg amniotic fluid
egg allantoic fluid
tissue culture cells
tissue culture supernatant
In this version we detail application of the QIAamp Viral RNA Mini Kit. Extraction involves lysis under highly denaturing conditions to inactivate RNases and isolate intact viral RNA.
Buffering conditions provide optimum binding of RNA to silica gel-based membrane of a mini spin column. RNA is eluted in RNase-free buffer or water by microcentrifugation. The extracted RNA can be used for a range of downstream genetic analyses.