SARS-CoV-2 Viral RNA Detection by qRT-PCR

Samples were collected with prewetted swabs in 1 mL of DMEM supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. Then, 140 μL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer's instructions with an elution volume of 150 μL. Tissues (up to 30 mg) were homogenized in RLT buffer and RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Viral RNA was detected by qRT-PCR [50 (link)]. Five μL RNA was tested with the Rotor-GeneTM probe kit (Qiagen) according to instructions of the manufacturer. Ten-fold dilutions of SARS-CoV-2 standards with known copy numbers were used to construct a standard curve.

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Yinda C.K., Port J.R., Bushmaker T., Offei Owusu I., Purushotham J.N., Avanzato V.A., Fischer R.J., Schulz J.E., Holbrook M.G., Hebner M.J., Rosenke R., Thomas T., Marzi A., Best S.M., de Wit E., Shaia C., van Doremalen N, & Munster V.J. (2021). K18-hACE2 mice develop respiratory disease resembling severe COVID-19. PLoS Pathogens, 17(1), e1009195.

Publication 2021

QIAamp Viral RNA Kit QIAcube HT automated system RNeasy kit Rotor-GeneTM probe kit

Buffer Dilutions Penicillin Sars cov 2 Streptomycin Tissues Viral rna

Corresponding Organization : National Institutes of Health

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Variable analysis

independent variables

Sample collection method (prewetted swabs in 1 mL of DMEM supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin)
RNA extraction method (QIAamp Viral RNA Kit using QIAcube HT automated system with an elution volume of 150 μL)
Tissue homogenization method (in RLT buffer)
RNA extraction method for tissues (RNeasy kit)

dependent variables

Viral RNA detection by qRT-PCR

control variables

Tissue sample size (up to 30 mg)
RNA sample volume for qRT-PCR (5 μL)
Use of SARS-CoV-2 standards with known copy numbers to construct a standard curve

positive controls

SARS-CoV-2 standards with known copy numbers

negative controls

None explicitly mentioned

Annotations

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