QIAamp Viral RNA Mini Kit (50)

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Publication protocol

Viral extractions were prepared in triplicate by manual QIAamp Viral RNA Mini Kit extraction (Qiagen) and automated extraction on the EZ1 Advanced XL (Qiagen) using the EZ1 Virus Mini Kit version 2.0, the MagNA Pure 96 (Roche), using the MagNA Pure 96 DNA and Viral NA small volume kit, the NucliSENS easyMag (BioMérieux) using the Specific B 2.0.1 protocol, and QIAsymphony (Qiagen) using the DSP Virus/Pathogen midi kit. Specific protocol B 2.0.1 was selected for use on the easyMag platform, as in our hands it has been found to give the highest viral RNA yields from this sample type. Sample extraction and elution volumes varied according to the extraction method, and details are displayed in Table 1. Qiagen’s inactivation buffer AVL, containing guanidinium thiocyanate, was used across all platforms because it is the standard inactivation medium capable of rendering highly pathogenic viruses safe to use at containment level 2, and an inert linear acrylamide (LA) carrier (Thermo Fisher Scien- tific, Waltham, MA) was added to each extraction as a coprecipitant to ensure maximum recovery of nucleic acids.25 For each concentration and method, two different AVL preparations were tested: AVL þ 5 mg LA carrier and AVL þ 5 mg LA carrier þ 1% (v/v) Triton X-100. Input concentrations were corrected for each extraction platform to provide an equal number of viral copies in the input material.
Proportional amounts of each sample were digested with DNase I (Life Technologies, Carlsbad, CA) following manufacturer’s instructions. The DNase I was inactivated, and the sample was cleaned up using a Zymo Clean and Concentrator column (Zymo Research, Irvine, CA) and eluted into 15-mL molecular grade water.

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Manufacturer protocol

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