Abstract
Gene 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.
Highlights
The RNAi Consortium (TRC) has developed genome-scale short hairpinRNA libraries targeting human and mouse genes [1], facilitating lentiviral-based RNA interference (RNAi)
We demonstrate that the conformation of the plasmid and its purity affect quantitative PCR (qPCR) efficiency, and that absolute quantification requires both the plasmid and viral cDNAs to be within an appropriate concentration range
NOTE: In the present study, target genes were neuropilin 1 (NRP1), plexin B1 (PLXNB1) and octamer-binding transcription factor 4 (OCT4, protein encoded by Pit-1 octamer unc-86 class 5 homeobox 1, abbreviated Pou5f1), and housekeeping genes were HPRT1, MAPK1 and UBC (Table S2)
Summary
The RNAi Consortium (TRC) has developed genome-scale short hairpin (sh)RNA libraries targeting human and mouse genes [1], facilitating lentiviral-based RNA interference (RNAi). In the context of functional titration, if a fluorescent reporter gene, e.g., green fluorescent protein [2], is transduced alongside the sequence of interest, the functional titer can be determined by flow cytometry. This requires a fluorescent marker and cannot distinguish single from multiple proviral DNA integration sites within the host genome. An alternative is to estimate the number of integrated proviral DNA copies per cell by quantitative (q)PCR [2]. Quantification of the expressed transgene by reverse transcription (RT)-qPCR instead of the proviral DNA copies gives a closer estimation of the transgene expression [2]
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