Viral total nucleic acid kit

Viral RNA was extracted from sera using the Viral Total Nucleic Acid Kit (Promega, Madison, WI) on a Maxwell 48 RSC instrument (Promega, Madison, WI). Viral RNA was isolated from homogenized tissues using the Maxwell 48 RSC Viral Total Nucleic Acid Purification Kit (Promega, Madison, WI) on a Maxwell 48 RSC instrument. Each tissue was homogenized using PBS supplemented with 20% FBS and penicillin/streptomycin and a tissue tearor variable speed homogenizer. Supernatant was clarified by centrifugation and the isolation was continued according to the Maxwell 48 RSC Viral Total Nucleic Acid Purification Kit protocol, and samples were eluted into 50 μl RNase free water. RNA was then quantified using quantitative RT-PCR. Viral load data from serum are expressed as vRNA copies/mL. Viral load data from tissues are expressed as vRNA copies/tissue.

vRNA was extracted from plasma using the Viral Total Nucleic Acid Kit (Promega, Madison, WI) on a Maxwell 48 RSC instrument (Promega, Madison, WI). RNA was then quantified using RT-qPCR. Viral load data from plasma are expressed as vRNA copies/ml.

Viral RNA was isolated from macaque blood samples as previously described [63 (link),64 (link)]. Briefly, plasma was isolated from EDTA-anticoagulated whole blood on the day of collection either using Ficoll density centrifugation for 30 minutes at 1860 x g if the blood was being processed for PBMC, or it was centrifuged in the blood tube at 1400 x g for 15 minutes. The plasma layer was removed and transferred to a sterile 15 ml conical and spun at 670 x g for an additional 8 minutes to remove any remaining cells. Viral RNA was extracted from a 300 μL plasma aliquot using the Viral Total Nucleic Acid Kit (Promega, Madison, WI) on a Maxwell 16 MDx or Maxwell RSC 48 instrument (Promega, Madison, WI).