A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR

Abstract

A B S T R A C TGene silencing techniques, including RNA interference methodologies, are widely used in reverse genetics to study the role of specific genes in biological processes. RNA interference has become easier to implement thanks to the RNAi Consortium (TRC), which has developed libraries of short hairpin RNA (shRNA) sequences in pseudotyped lentiviral particles capable of targeting most genes in the human and mouse genomes. However, a problem is the lack of a simple method to titrate the homemade lentiviral particle product, making it difficult to optimize and standardize shRNA experiments. Here we provide a guide describing a quick, non-laborious and reliable method for the titration of TRC pseudotyped lentiviral particles that is based on the detection and measurement of viral RNA using quantitative PCR. Our data demonstrate that purified linearized shRNA plasmids represent more suitable standards than circular or unpurified linearized plasmids. We also show that for precise absolute quantification, it is important to determine suitable plasmid and viral cDNA concentrations in order to find the linear range for quantification, as well as to reduce inhibition and primer dimer amplification. Finally, we show that the lentivirus concentration impacts the level of knockdown in transduced cells. Primers utilized in this non-functional titration can potentially be applied to functional titration of proviral DNA copies or transgene expression, overcoming problems arising from the absence of fluorescent reporter genes in TRC plasmids.

Establishment of a pseudovirus neutralization assay based on SARS-CoV-2 S protein incorporated into lentiviral particles

“…VSV-G pseudovirus was used as a control. Virus titers were determined by calculating the concentrations of viral RNA genomes using quantitative RT-PCR and measuring RLU based on a Luc assay [18] . The HEK-293T, established HEK-293T cells stably expressing human ACE2 (293T-ACE2) and HuH7 were used to test the correlation between ACE2 expression and pseudovirus susceptibility.…”