A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR
Main Article Content
Keywords
lentiviral particle titre, absolute quantification, shRNA, The RNAi Consortium, RT-qPCR
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 optimise and standardise 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 linearised shRNA plasmids represent more suitable standards than circular or unpurified linearised 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 utilised 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.
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References
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27. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. doi:10.1006/meth.2001.1262
28. Saito T, Sætrom P (2012) Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments. Silence 3: 3. doi:10.1186/1758-907X-3-3
29. Taxman DJ, Livingstone LR, Zhang J, Conti BJ, Iocca HA, Williams KL, et al. (2006) Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol 6: 7. doi:10.1186/1472-6750-6-7
30. Böcker W, Rossmann O, Docheva D, Malterer G, Mutschler W, Schieker M (2007) Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells. J Gene Med 9: 585–95. doi:10.1002/jgm.1049
2. Lizée G, Aerts JL, Gonzales MI, Chinnasamy N, Morgan RA, Topalian SL (2003) Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther 14: 497–507. doi:10.1089/104303403764539387
3. Gay V, Moreau K, Hong SS, Ronfort C (2012) Quantification of HIV-based lentiviral vectors: Influence of several cell type parameters on vector infectivity. Arch Virol 157: 217–223. doi:10.1007/s00705-011-1150-5
4. Geraerts M, Willems S, Baekelandt V, Debyser Z, Gijsbers R (2006) Comparison of lentiviral vector titration methods. BMC Biotechnol 6: 34. doi:10.1186/1472-6750-6-34
5. Towers GJ, Stockholm D, Labrousse-Najburg V, Carlier F, Danos O, Pages JC (1999) One step screening of retroviral producer clones by real time quantitative PCR. J Gene Med 1: 352–359. doi:10.1002/(SICI)1521-2254(199909/10)1:5<352::AID-JGM57>3.0.CO;2-I
6. Sastry L, Johnson T, Hobson MJ, Smucker B, Cornetta K (2002) Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther 9: 1155–1162. doi:10.1038/sj.gt.3301731
7. Scherr M, Battmer K, Blömer U, Ganser A, Grez M (2001) Quantitative determination of lentiviral vector particle numbers by real-time PCR. Biotechniques 31: 520–526.
8. Sun C, Li Y, Taylor SE, Mao X, Wilkinson MC, Fernig DG (2015) HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors. PeerJ 3: e1060. doi:10.7717/peerj.1060
9. Baxter MA, Camarasa MV, Bates N, Small F, Murray P, Edgar D, et al. (2009) Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines. Stem Cell Res 3: 28–38. doi:10.1016/j.scr.2009.03.002
10. Dhanasekaran S, Doherty TM, Kenneth J, Group TBTS (2010) Comparison of different standards for real-time PCR-based absolute quantification. J Immunol Methods 354: 34–39. doi:10.1016/j.jim.2010.01.004
11. Logan AC, Nightingale SJ, Haas DL, Cho GJ, Pepper KA, Kohn DB (2004) Factors influencing the titer and infectivity of lentiviral vectors. Hum Gene Ther 15: 976–988. doi:10.1089/hum.2004.15.976
12. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9: 654–9. doi:10.1038/ncb1596
13. Ikeda Y, Takeuchi Y, Martin F, Cosset FL, Mitrophanous K, Collins M (2003) Continuous high-titer HIV-1 vector production. Nat Biotechnol 21: 569–572. doi:10.1038/nbt815
14. Wacker MJ, Godard MP (2005) Analysis of one-step and two-step real-time RT-PCR using superscript III. J Biomol Tech 16: 266–271. doi:16/3/266 [pii]
15. Bustin SA, Nolan T (2004) Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 15: 155–166. doi:15/3/155 [pii]
16. StÃ¥hlberg A, Kubista M, Pfaffl M (2004) Comparison of reverse transcriptases in gene expression analysis. Clin Chem 50: 1678–1680. doi:10.1373/clinchem.2004.035469
17. Levesque-Sergerie JP, Duquette M, Thibault C, Delbecchi L, Bissonnette N (2007) Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR. BMC Mol Biol 8: 93. doi:10.1186/1471-2199-8-93
18. Fleige S, Pfaffl MW (2006) RNA integrity and the effect on the real-time\rqRT-PCR performance. Mol Aspects Med 27: 126–139. doi:10.1016/j.mam.2005.12.003
19. Chen YP, Evans J, Hamilton M, Feidlaufer M (2007) The influence of RNA integrity on the detection of honey bee viruses: molecular assessment of different sample storage methods. J ofApiCa1ri Res 46: 81–87. doi:10.3896/IBRA.1.46.2.03
20. Whelan JA, Russell NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods 278: 261–269. doi:10.1016/S0022-1759(03)00223-0
21. Lee C, Kim J, Shin SG, Hwang S (2006) Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123: 273–280. doi:10.1016/j.jbiotec.2005.11.014
22. Tang H, Kuhen KL, Wong-Staal F (1999) Lentivirus replication and regulation. Annu Rev Genet 33: 133–170. doi:10.1146/annurev.genet.33.1.133
23. StÃ¥hlberg A, HÃ¥kansson J, Xian X, Semb H, Kubista M (2004) Properties of the Reverse Transcription Reaction in mRNA Quantification. Clin Chem 50: 509–515. doi:10.1373/clinchem.2003.026161
24. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13: 134. doi:10.1186/1471-2105-13-134
25. Svec D, Tichopad A, Novosadova V, Pfaffl MW, Kubista M (2015) How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif 3: 9–16. doi:10.1016/j.bdq.2015.01.005
26. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8: R19. doi:10.1186/gb-2007-8-2-r19
27. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. doi:10.1006/meth.2001.1262
28. Saito T, Sætrom P (2012) Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments. Silence 3: 3. doi:10.1186/1758-907X-3-3
29. Taxman DJ, Livingstone LR, Zhang J, Conti BJ, Iocca HA, Williams KL, et al. (2006) Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol 6: 7. doi:10.1186/1472-6750-6-7
30. Böcker W, Rossmann O, Docheva D, Malterer G, Mutschler W, Schieker M (2007) Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells. J Gene Med 9: 585–95. doi:10.1002/jgm.1049
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Published
Oct 4, 2016
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Mournetas V, Pereira SM, Fernig DG, Murray P. A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR. J Biol Methods [Internet]. 2016 Oct. 4 [cited 2024 Apr. 18];3(4):e55. Available from: https://jbmethods.org/index.php/jbm/article/view/142
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Author Biographies
Sofia M. Pereira, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, L697ZB Liverpool, United Kingdom
Department of Cellular and Molecular PhysiologyDavid G. Fernig, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, L697ZB Liverpool, United Kingdom
Department of BiochemistryPatricia Murray, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, L697ZB Liverpool, United Kingdom
Department of Cellular and Molecular Physiology
