Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation.
The methodological and logistic benefits gained from assessing in vitro antiretroviral replication using one-tube real-time RT-PCR procedures are currently diminished by a continuing need for prior RNA isolation. We now report a simple and inexpensive modification of a commercially available one-tube RT-PCR assay, consisting of detergent-based virus lysis in the presence of a ribonuclease inhibitor, which can be used to directly quantify retroviral RNA levels in culture supernatant. This approach circumvents the potential loss of RNA inherent to RNA-isolation procedures based on prior extraction and demonstrates a dynamic range of at least 4 logs. Using in vitro culture systems incorporating either HIV-1 or FIV, we show that this ability to isolate retroviral RNA directly during the RT-PCR process can provide an equivalent alternative to one of the more time and resource-consuming steps in quantifying in vitro retroviral RNA levels.
Bisset
L R
LR
Swiss National Center for Retroviruses, University of Zürich, Gloriastrasse 30, CH-8028 Zürich, Switzerland.
Bosbach
S
S
Tomasik
Z
Z
Lutz
H
H
Schüpbach
J
J
Böni
J
J
D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D002415:Cats; D002460:Cell Line; D003365:Costs and Cost Analysis; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015683:Gene Products, gag / Q000235:genetics; D015684:Gene Products, pol / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D016089:Immunodeficiency Virus, Feline / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification; D012367:RNA, Viral / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000187:drug effects; D015215:Zidovudine / Q000494:pharmacology
D019380:Anti-HIV Agents; D000998:Antiviral Agents; D015683:Gene Products, gag; D015684:Gene Products, pol; D012333:RNA, Messenger; D012367:RNA, Viral; D015215:Zidovudine