Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs

Marta Massanella, Sara Gianella, Steven Lada, Douglas Richman, Matthew Strain
2015 Bio-protocol  
Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood
more » ... ononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR). For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to protocol "Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma" (Vargas-Meneses et al., 2015). Materials and Reagents A. Cell processing and flow cytometry 1. 1x Dulbecco's phosphate buffered saline (DPBS) (Corning, catalog number: 21-031-CV) 2. RPMI 1640 medium (Life Technologies, Gibco ® , catalog number: 11875-093) 3. GemCell U.S. origin fetal bovine serum (FBS) (Gemini Bio Products, catalog number: 100-500) 4. 37% formaldehyde solution (Sigma-Aldrich, catalog number: F8775-500ML) 5. Sodium azide Reagents Plus (NaN3) (Sigma-Aldrich, catalog number: S2002-25G) 6. Antibodies a. CD3-APC (BD, catalog number: 340440) b. CD4-FITC (BD, catalog number: 347413) c. CD45-PerCP-Cy5.5 (BD, catalog number: 340953) 7. AllPrep DNA/RNA Mini Kit (QIAGEN, catalog number: 80204) 8. Staining flow cytometry buffer (staining buffer) (see Recipes) 9. 1% formaldehyde (1% FA) buffer (see Recipes) 10. RLT plus buffer + β-ME (see Recipes) (included in the AllPrep DNA/RNA Mini Kit)
doi:10.21769/bioprotoc.1492 fatcat:bbipnd7n4nao7erj56v7hdszf4