Reverse Transcriptase Activity as a Marker for Virus-Like Particle Production

Résumé

Source: Scarborough, R. J., et al. Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy. J. Vis. Exp. (2016).

This video demonstrates a reverse transcriptase (RT) assay to assess the effect of a test RNA on virus-like particle (VLP) production. Cells are co-transfected with plasmids, and RT activity is measured as a quantitative indicator of VLP output. A decrease in RT activity reflects suppression of viral gene expression by the test RNA.

Protocole

1. Viral Production Assay

1. Remove the 24-well cell culture plates from the incubator and gently swirl the plates inside the biosafety level 3 (BSL3) cell culture hood. Transfer 150 µl of supernatant from each well to a corresponding well in a 96-well flat-bottom plate, which will be used to quantify HIV-1 production.

Note: Common viral quantification assays include measuring the expression of the human immunodeficiency virus (HIV)-1 capsid protein (p24) by enzyme-linked immunosorbent assay (ELISA), quantifying viral RNA by reverse transcription polymerase chain reaction (RT-PCR), and measuring the activity of the HIV-1 RT enzyme....

Résultats

FIGURE 1: Effect of test siRNAs on HIV-1 production. (A) Design of siRNAs with different lengths and symmetries. A conserved sequence in HIV-1 RNA (1498 target site) was used to design symmetrical and asymmetrical siRNAs (si1498-) with differe.......

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
Cell culture plates, 96 well, 24 well, 6 well.	Corning	353075, 353047, 353043
[³²P]dTTP	Perkin Elmer	BLU505H
Poly(A) RNA template	Sigma-Aldrich	10108626001
Oligo(dT)12-18 DNA primer	Thermo Fisher	18418-012
DEAE filtermat paper	Perkin Elmer	1450-522
Microplate scintillation counter	Perkin Elmer	1450-024
Lysis buffer tablets	Roche	4693159001, 4906837001	protease and phosphatase inhibitors