RNA from these reference viral isolates was extracted using the QIAamp Viral RNA mini kit, according to manufacturer's instructions (Qiagen, Valencia, CA), eluted in 60 ml of elution buffer and then diluted 1:1000. A DBS panel was prepared by mixing 60 ml of each virus isolate with 240 ml of seronegative donor whole blood and then spotting 50 ml of this spiked donor blood onto 903 filter paper. Using the protocol described below, this dried blood spot panel was extracted and run in the rtLC DBS assay. Preparation of Standard Curve. To ensure uniformity and reproducibility in the DBS preparation and extraction process, we prepared customized DBS standards with known viral copies. An HIV-1 clade B infected patient isolate with known viral load was selected and spiked into HIV negative donor whole blood to prepare DBS in five fold serial dilutions. A series of 5 independent extractions were performed and quantified using the rtLC DBS assay in a total of 25 independent runs.