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. 2016 Oct 4;3(4):e55.
doi: 10.14440/jbm.2016.142. eCollection 2016.

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

Virginie Mournetas  1   2   3 , Sofia Melo Pereira  1 , David G Fernig  2 , Patricia Murray  1
Affiliations

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

Virginie Mournetas et al. J Biol Methods. .

Erratum in

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.

Keywords: RT-qPCR; absolute quantification; lentiviral particle titer; shRNA; the RNAi Consortium.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1.
Figure 1.
From plasmid amplification to pseudotyped lentiviral particle quantification. Main steps in the timeline of pseudotyped lentiviral particle production, starting with plasmid amplification and ending with pseudotyped lentiviral particle absolute quantification.
Figure 2.
Figure 2.
qPCR efficiencies and dynamic range of the standard curves. A. Efficiency curves of circular (red), linearized (orange) and purified linearized (blue): shOCT4-79 (left panel) and shControl plasmids (right panel). Linear regression curves are given with their correlation coefficient R2. The purified linearized plasmids give a slope of -4.01 and -4.18 respectively, which are the closest to -3.32, the ideal slope resulting in an amplification efficiency of 100%. Examples of amplification curves (B), melting peaks (C) and agarose gel of qPCR products from the shSERPINE1 (D) purified linearized standard. The tested plasmid concentration range is represented: the highest in orange, the intermediate in green and the lowest in red. E. Efficiency curves of purified linearized shSERPINE1 standard with the two highest plasmid concentrations (purple), without the highest concentration of 5 ng/reaction (green) or without the both highest tested concentrations of 5 and 1 ng/reaction (blue). Linear regression curves are given with their correlation coefficient R2. The blue curve gives a slope of -3.52 which is the closest to -3.32, the ideal slope resulting in an amplification efficiency of 100%.
Figure 3.
Figure 3.
shRNA pseudotyped lentiviral particle absolute quantification. A-C. Viral cDNA quantification by qPCR: examples of amplification curves (A), melting peaks (B) and agarose gel of qPCR products from the shPLXNB1 viral cDNA (C). The viral cDNA concentration in green was the highest tested and in orange the lowest. The NRT is represented in red. D-F. cDNA amplification from non-transfected producer cells: amplification curves (D), melt peaks (E) and agarose gel of qPCR products from amplified cDNA of purified secreted RNA of HEK-293TN cells in orange, an example of amplified shOCT4-79 plasmid in green and NTC in red (F). G-H. Effect of the lowest and highest viral cDNA concentrations on shRNA pseudotyped lentiviral particle absolute quantification: comparisons of shRNA lentiviral particle absolute quantification using intermediate (medium) and the lowest (G) or the highest (H) viral cDNA concentrations (pseudotyped lentiviral particles/µl of supernatant, mean ± SEM; shNRP1=shNRP1-24; shOCT4=shOCT4-79; shPLXNB1=shPLXNB1-35).
Figure 4.
Figure 4.
Effect of shRNA lentiviral concentration on knockdown efficiency. A. Relative gene expression results measuring the knockdown efficiency of NRP1, PLXNB1 and OCT4 with two different shRNA concentrations C1 (18 pg viral RNA/cell) and C2 (36 pg viral RNA/cell. Only C2 was used for shControl ( normalisation done against untransduced hESCs; mean ± SEM; One sample t-test, *0.01 < P < 0.05, *** P < 0.001, N.S.: not significant, no star means n < 3). B-E. Correlations (logarithmic fitting curves) between the amount of shRNA lentiviral particles (absolute number) and the knockdown efficiency (%) of NRP1 (purple), PLXNB1 (red) and OCT4 (orange) together or separately.
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