- Email: [email protected]
Quantitation of MLV-based retroviral vectors using real-time RT-PCR
Journal of Virological Methods 119 (2004) 115–119
Quantitation of MLV-based retroviral vectors using real-time RT-PCR M. Carmo a , C. Peixoto a , A.S. Coroadinha a , P.M. Alves a , P.E. Cruz a,b,c , M.J.T. Carrondo a,d,∗ a IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal ECBio, Lab 4.11, Ed. ITQB, Apartado 98, P-2781-901 Oeiras, Portugal c Universidade Atlˆ antica, Antiga Fábrica da Pólvora, P-2745-615 Barcarena, Portugal Laboratório de Engenharia Bioqu´ımica, FCT/UNL, P-2825 Monte da Caparica, Portugal b
d
Received 5 December 2003; received in revised form 16 March 2004; accepted 18 March 2004
Abstract Murine leukaemia virus-based vectors quantitation is a time consuming process that can take up to five days. In order to reduce this time a real-time RT-PCR was developed. This method quantifies vectors without an RNA extraction step, using AMV reverse transcriptase and LightCycler technology. Besides a low quantitation time, this method has the advantages of using a plasmid DNA standard curve with good reproducibility, and of having a high sensitivity (3 × 102 particles/l) as well as an excellent intra- and inter-assay reproducibility. Although the method described quantifies vector particles with RNA whether these particles are infectious or not, it is possible to use it to determine infectious particles concentration after the establishment of a correlation between particles with RNA and infectious particles, for a given set of conditions. This method can also be used to study vector stability by comparison of infectious particles, total particles and particles with RNA. © 2004 Elsevier B.V. All rights reserved. Keywords: Retroviral vectors; Quantitation; Real-time RT-PCR
1. Introduction Retroviral vectors are one of the most widely used gene delivery vehicles in clinical gene therapy protocols, especially murine leukaemia virus (MLV)-based ones (Zhao et al., 2000; McTaggart and Al-Rubeai, 2002). The high potential of this type of vectors is due to their broad range of hosts, the transferred genes are integrated stably into chromosomes of the host and the transferred gene is transmitted without rearrangements (Andreadis et al., 1999). Despite all the advantages of retroviral vectors there are some problems: low transduction efficiencies; cell division is required for integration into the genome of the target cell; low viral titters production; and low stability (Andreadis et al., 1999; Cruz et al., 2000). ∗ Corresponding author. Tel.: +351-21-442-77-87; fax: +351-21-442-11-61. E-mail address: [email protected] (M.J.T. Carrondo).
0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.03.007
Several methods can be used to quantify retroviral vectors: titration assays (Forestell et al., 1995; Lee et al., 1996) and reverse transcriptase activity assays (Kwon et al., 2003) to quantify infectious particles; and electron microscopy to quantify total particles (Higashikawa and Chang, 2001). Other methods exist that are semi-quantitative such as RT-PCR (Heinemeyer et al., 1997) and immunoblotting (Muriaux et al., 2002). Some new methods have been developed from the conventional PCR method such as real-time PCR that with on-line and real-time monitoring of fluorescent-labelled PCR products enables a rapid and quantitative determination of sample DNA concentration, without the need of gel products detection (Rasmussen, 2001; Müller and Wirth, 2002). This quantitation is achieved using software that calculates the copy number of the templates in respect to a standard curve generated in parallel. Slopes of the fluorescence accumulation curves are calculated during the log-linear phase of amplification so that the quantitation is based on kinetic analyses not relying
116
M. Carmo et al. / Journal of Virological Methods 119 (2004) 115–119
on end-point PCR products (Rasmussen, 2001; Saha et al., 2001). In order to reduce the time needed for MLV-based vector quantitation, a real-time RT-PCR method was developed. This method quantifies vectors without an RNA extraction step using avian myeloblastosis virus (AMV) reverse transcriptase and LightCycler technology.
2. Materials and methods 2.1. Production of retroviral vectors Retroviral vectors were obtained from supernatant of human TE FLY A7 packaging cell line, derived from TE 671 cells (ECACC no. 89071904) transformed with the plasmid pMFGSnlsLacZ (Pizzato et al., 2001) that produces murine leukaemia virus-based vectors; this cellular line was kindly provided by Dr. Otto Merten from Généthon, France. These cells were cultured in DMEM medium (Gibco, Paisley, UK) supplemented to have 4.5 g/L of glucose (Merck, Darmstadt, Germany), 6 mM of glutamine (Gibco) and 5% FBS (Gibco). After three days of culture the medium was replaced and vectors were produced during 24 h. 2.2. Choice of standard and primers The plasmid pSIR (Clontech, Palo Alto, USA) was chosen as standard to quantify retroviral RNA; pSIR is a plasmid derived from Moloney murine leukaemia virus. Forward and reverse primer sequences specific for MLV were located within the LTR region and consisted of 5 -ATT GAC TGA GTC GCC CGG-3 and 5 -AGC GAG ACC ACA AGT CGG AT-3 , respectively. 2.3. Pre-treatment of supernatants Cell culture supernatants were filtered with 0.45 m pore filters to remove cells and cellular debris and incubated at 75 ◦ C for 10 min to release RNA. In order to destroy DNA derived from lysed cells 2 l DNaseI (1 U/l, Sigma, Steinheim, Germany) and 4 l 25mM MgCl2 were incubated with 100 l of filtered supernatant for 30 min at room temperature. 2.4. cDNA synthesis cDNA synthesis was performed using first strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). DNase I pre-treated supernatants were incubated for 10 min at 75 ◦ C. Following this, 11.5 l of cDNA synthesis mix was prepared to the indicated end-concentrations: 2 l of reaction buffer (1×), 4 l MgCl2 (5 mM), 2 l dNTP (1 mM), 1.7 reverse primer (1 M), 1 l RNase inhibitor (50 U) and 0.8 l AMV reverse transcriptase (20 U). The
8.5 l of supernatant was added to the cDNA synthesis mix, incubated for 10 min at 25 ◦ C and then incubated for 1 h at 42 ◦ C. A subsequent heat inactivation step of 5 min at 99 ◦ C was performed and the final cDNA was stored at −20 ◦ C. 2.5. Real-time PCR For LightCycler reaction a mastermix of the following reaction components was prepared to the indicated end-concentrations: 2 l LightCycler master (Fast start DNA master SYBR Green I; Roche Diagnostics), 3.2 l MgCl2 (4 mM), 0.6 l forward primer (0.5 M), 0.86 l reverse primer (0.5 M) and 3.34 l PCR grade water. LightCycler master mix was distributed into the LightCycler capillaries (10 l each) and 10 l of cDNA or pSIR standard were added as PCR template. Capillaries were closed and placed into the light cycler rotor. The following LightCycler run protocol was used: denaturation program (95 ◦ C for 10 min); amplification and quantitation program repeated 45 times (60 ◦ C for 10 min; 72 ◦ C for 10 min with a single fluorescent measurement); melting curve program (65–95 ◦ C for 10 min with continuous fluorescent measurement); and finally a cooling step to 40 ◦ C. For visualisation, 10 l of the reaction products were separated by gel electrophoresis on 2% agarose gel in TBE buffer containing ethidium bromide. In case of successful amplification a product around 73 bp in length indicated the presence of murine C-type retroviral nucleic acids. 2.6. Quantitation of infectious particles For determination of the concentration of retroviral infectious particles, target cells HCT 116 (ATCC no. CCL-247) were seeded in 96-well, flat-bottomed plates (Starstedt, Newton, USA) at a density of 5 × 104 cells/cm2 and incubated during 24 h. Infections were then carried out by replacing the medium with 20 l of dilutions (10−1 to 10−4 ) of viral supernatants in DMEM medium containing 8 g/ml of polybrene (Sigma) followed by incubation at 37 ◦ C for 4 h. After this time 180 l of fresh medium was added to the plate, and infected cells were incubated for 1 day. Then, the medium of each well was aspirated and washed with 100 l of PBS. The 100 l of fixing solution, with 0.75% formaldehyde 37% (Merck) and 5.1% of glutaraldehyde (Sigma) in PBS, was added to each well and left for 2 min before aspiration; then each well was washed with 100 l of PBS. Next, 100 l of the dye consisting of 5 mM K3 Fe8 (CN)6 (Merck), 5 mM K4 Fe(CN)6 (Merck), 1 mM MgCl2 (Merck) and 200 mg/ml X-gal (Stratagene, La Jolla, USA) in DMF (Riedel deHaën, Seelze, Germany) was added to each well, and the LacZ-positive (blue) cells were counted after 24 h of incubation at 37 ◦ C. For each sample three dilution sets were performed.
M. Carmo et al. / Journal of Virological Methods 119 (2004) 115–119
117
3. Results
Table 1 Method efficiencya
3.1. Standard curve validation
Slopestandard
Estandard
Slopesamples
Esamples
Estandard − Esamples
−3.38 −3.44 −3.42
1.98 1.95 1.96
−3.32 −3.39 −3.32
2.00 1.97 2.00
0.03 0.02 0.04
In LightCycler real-time RT-PCR a standard curve must be used to quantify DNA. A DNA plasmid (pSIR) with MLV sequence was chosen as standard in this method. After a few optimisations (plasmid purification by precipitation with ammonium acetate and quantitation using GeneQuantTM , Amersham, UK) a standard curve with good efficiency and reproducibility was obtained. The efficiency is shown by the slope of the standard curve with the log of the initial template copy number plotted horizontally and the cycle number at the crossing point plotted vertically. In this case the slope obtained was around 3.4 (must be between 3.3 and 3.9), the reproducibility was seen calculating the variation coefficient between three repetitions in a row and this was less than 1%. 3.2. Confirmation of primer specificity Specificity of real-time RT-PCR products was verified by agarose gel electrophoresis (2%) and resulted in a single product around 70 bp, as desired (Fig. 1). In addition a LightCycler melting curve was performed which resulted in single product with specific melting temperature of 85 ◦ C. 3.3. RNA quantitation After standard curve optimisation and primer specificity confirmation a test must be done to evaluate the method efficiency amplifying retroviral vector RNA. In order to have high method efficiency, the difference between efficiency of the standard curve and efficiency of the RNA sample curve must be lower than 0.05 and the amount of PCR product present at the crossing point must be the same for standards and samples (Rasmussen, 2001), with the efficiency being calculated as E = 10(−1/Slope) . The test was repeated three
a Comparison between efficiency of the standard curve (E standard ) and efficiency of an RNA samples curve (Esamples ).
Table 2 Intra- and inter-assay variability, standard deviation and coefficient of variability (CV) for three experiments in a row Average RNA copy (l)
S.D.
CV intra-assay (%)
CV inter-assay (%)
4.33 × 103 5.31 × 103 4.50 × 103
8.30 × 101 6.66 × 101 1.09 × 102
2 1 2
3
times in a row and the difference of efficiency was always lower than 0.05 and the amount of PCR product present at the crossing point was the same for standards and samples. These results are shown in Table 1. 3.4. Intra- and inter-assays To confirm the accuracy and reproducibility of real-time RT-PCR the intra-assay variation was determined in three repeats within a LightCycler run. Inter-assay variation was investigated in three different experimental runs performed on 3 days using three different master mixes of LightCycler Fast start DNA master SYBR green I kit (Roche diagnostics). Test variation was 2% in intra-test experiments and 3% in inter-test experiments, as shown in Table 2. 3.5. Sensitivity For evaluation of assay sensitivity serial dilutions of supernatants with retroviral vectors were subjected to real-time RT-PCR. The results show that with this method retroviral vectors can be amplified from a minimum concentration of 3 × 102 RNA copies/l. 3.6. RNA concentration versus infectious vectors To understand the relationship between vectors with RNA and infectious vectors, three samples of vectors were quantified by real-time RT-PCR and titre assay in the same day. The results are presented in Table 3. It can be noticed that the Table 3 Comparison between vectors with RNA (RNA V) and infectious vectors (IV)
Fig. 1. Specificity of real-time RT-PCR. M: PCR markers (Promega, Madison, USA) 1000, 750, 500, 300, 150, 50 bp; lanes 1–6: pSIR plasmid; lane 7: H2 O control; lane 8–11: supernatant sample.
RNA V
IV
RNA V/IV
1.38 × 109 1.80 × 109 1.73 × 109
1.52 × 107 1.95 × 107 2.00 × 107
91 92 89
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ratio of vectors with RNA and infectious vectors is around 90 with a coefficient of variability of 2%, meaning that the majority of the vectors that contain RNA are not infectious.
4. Discussion We describe a real-time RT-PCR method for quantitation of retroviral vector particles with RNA. The presented protocol can be used for any MLV-based retroviral vectors since the quantitation is performed with primers specific for the LTR region. Real-time RT-PCR is a highly sensitive method that allows quantitation of rare transcripts. It is also easy to perform, provides necessary accuracy and produces reliable and rapid quantitation results (Pfaffl, 2001). With this method two quantitation types are possible: a relative quantitation based on the relative expression of a target gene versus a reference gene; and an absolute quantitation based either on an internal or an external calibration curve (Rasmussen, 2001). The method uses plasmid DNA as standard; this has the advantage of being a standard easy to quantify and stable. Some methods exist that use real-time PCR to quantify retroviral vector particles with culture supernatant containing retroviral particles as standard. But retroviral vectors are very unstable having a half-life of 5–8 h at 37 ◦ C (Andreadis et al., 1999; Higashikawa and Chang, 2001; McTaggart and Al-Rubeai, 2002) and are sensitive to freezing and thawing, making reproducibility very difficult (Lee et al., 1996; Higashikawa and Chang, 2001). Another problem with these methods is that the standard supernatants are monitored overtime by their infectious particle titre, not by their RNA copy content, since this is used as standard. Another advantage of the new method is that it does not need an RNA extraction step. A heat step at 75 ◦ C is used to destroy vector particles, RNA becoming free in solution. Furthermore, this method presents low variability between the intra-assay tests (2%), ensuring a good precision, and low variability between the inter-assay tests (3%) meaning that the method has good reproducibility. Normally, to quantify infectious retroviral vectors a titre assay is used that take 5 days for completion (Forestell et al., 1995; Lee et al., 1996), whereas the method presented here has the ability to quantify vectors in a few hours. However, the production of defective particles is a problem concerning retroviral vectors production (Forestell et al., 1995; Higashikawa and Chang, 2001). These particles can be produced without envelope, without RNA or with RNA but not being infectious. The method described quantifies all vector particles containing RNA. The establishment of a correlation between vectors with RNA and infectious vectors for each specific situation can solve this problem, since this ratio can be applied to quantitation. A test was made to calculate the ratio between vectors with RNA and infectious particles; this ratio was found to be around 90 with a variability of 2%, for the standard production conditions defined in this work. So to quantify infectious vectors through this method
the RNA copy number obtained must be divided by this ratio. The new method is also useful to study vector stability by comparison of infectious vectors with vectors with RNA and total vectors. The smaller the differences between these three types of vectors the more stable are the vectors produced. With minor modifications, this method also can be used to study other retrovirus, even true RNA viral families. In conclusion, the retroviral vector quantitation method described above shows a very good reproducibility in interand intra-assays. By using a plasmid as a standard, it is ensured that the results are consistent between different production runs. The sensitivity of the method is low enough to allow the characterisation of most culture supernatants. Moreover, this method can be used for the determination of infectious particles under standard production conditions, thus allowing a faster quantitation of the vectors produced. Finally, it is possible, by including this method in a broader vector analysis strategy, to have a better characterisation of the produced supernatant and to study the effects of vector inactivation during downstream processing and storage as well as in the quality analysis of clinical-grade material.
Acknowledgements The authors acknowledge the financial support received from the European commission (QLK3-CT-2002-01949) and the Fundação para a Ciˆencia e Tecnologia, Portugal (POCTI SFRHBD31282000 and POCTI/1999/BIO/35695).
References Andreadis, S.T., Roth, C.M., Le Doux, J.M., Morgan, J.R., Yarmush, M.L., 1999. Large-scale processing of recombinant retroviruses for gene therapy. Biotechnol. Prog. 15 (1), 1–11. Cruz, P.E., Almeida, J.S., Murphy, P.N., Moreira, J.L., Carrondo, M.J., 2000. Modeling retrovirus production for gene therapy. 1. Determination of optimal bioreaction mode and harvest strategy. Biotechnol. Prog. 16 (2), 213–221. Forestell, S.P., Bohnlein, E., Rigg, R.J., 1995. Retroviral end-point titer is not predictive of gene transfer efficiency: implications for vector production. Gene Ther. 2 (10), 723–730. Heinemeyer, T., Klingenhoff, A., Hansen, W., Schumacher, L., Hauser, H., Wirth, M., 1997. A sensitive method for the detection of murine C-type retroviruses. J. Virol. Meth. 63 (1–2), 155–165. Higashikawa, F., Chang, L., 2001. Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280 (1), 124–131. Kwon, Y.J., Hung, G., Anderson, W.F., Peng, C.A., Yu, H., 2003. Determination of infectious retrovirus concentration from colony-forming assay with quantitative analysis. J. Virol. 77 (10), 5712–5720. Lee, S.G., Kim, S., Robbins, P.D., Kim, B.G., 1996. Optimization of environmental factors for the production and handling of recombinant retrovirus. Appl. Microbiol. Biotechnol. 45 (4), 477–483. McTaggart, S., Al-Rubeai, M., 2002. Retroviral vectors for human gene delivery. Biotechnol. Adv. 20, 1–31. Müller, K., Wirth, M., 2002. Real-time RT-PCR detection of retroviral contaminations of cells and cell lines. Cytotechnology 38 (1–3), 147– 153.
M. Carmo et al. / Journal of Virological Methods 119 (2004) 115–119 Muriaux, D., Mirro, J., Nagashima, K., Harvin, D., Rein, A., 2002. Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes. J. Virol. 76 (22), 11405–11413. Pfaffl, M.W., 2001. A new mathematical model for relative quantitation in real-time RT-PCR. Nucl. Acids Res. 29 (9), 2002–2007. Pizzato, M., Merten, O.W., Blair, E.D., Takeuchi, Y., 2001. Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors. Gene Ther. 8, 737–745.
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Rasmussen, R., 2001. Quantitation on the LightCycler. In: Meuer, S., Wittwer, C., Nakagawara, K.-i. (Eds.), Rapid Cycle Real-time PCR—Methods and Applications. Springer-Verlag, pp. 21–34. Saha, B.K., Tian, B., Bucy, R.P., 2001. Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe. J. Virol. Meth. 93 (1–2), 33–42. Zhao, Y., Low, W., Collins, M.K., 2000. Improved safety and titre of murine leukaemia virus (MLV)-based retroviral vectors. Gene Ther. 7 (4), 300–305.
Quantitation of MLV-based retroviral vectors using real-time RT-PCR M. Carmo a , C. Peixoto a , A.S. Coroadinha a , P.M. Alves a , P.E. Cruz a,b,c , M.J.T. Carrondo a,d,∗ a IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal ECBio, Lab 4.11, Ed. ITQB, Apartado 98, P-2781-901 Oeiras, Portugal c Universidade Atlˆ antica, Antiga Fábrica da Pólvora, P-2745-615 Barcarena, Portugal Laboratório de Engenharia Bioqu´ımica, FCT/UNL, P-2825 Monte da Caparica, Portugal b
d
Received 5 December 2003; received in revised form 16 March 2004; accepted 18 March 2004
Abstract Murine leukaemia virus-based vectors quantitation is a time consuming process that can take up to five days. In order to reduce this time a real-time RT-PCR was developed. This method quantifies vectors without an RNA extraction step, using AMV reverse transcriptase and LightCycler technology. Besides a low quantitation time, this method has the advantages of using a plasmid DNA standard curve with good reproducibility, and of having a high sensitivity (3 × 102 particles/l) as well as an excellent intra- and inter-assay reproducibility. Although the method described quantifies vector particles with RNA whether these particles are infectious or not, it is possible to use it to determine infectious particles concentration after the establishment of a correlation between particles with RNA and infectious particles, for a given set of conditions. This method can also be used to study vector stability by comparison of infectious particles, total particles and particles with RNA. © 2004 Elsevier B.V. All rights reserved. Keywords: Retroviral vectors; Quantitation; Real-time RT-PCR
1. Introduction Retroviral vectors are one of the most widely used gene delivery vehicles in clinical gene therapy protocols, especially murine leukaemia virus (MLV)-based ones (Zhao et al., 2000; McTaggart and Al-Rubeai, 2002). The high potential of this type of vectors is due to their broad range of hosts, the transferred genes are integrated stably into chromosomes of the host and the transferred gene is transmitted without rearrangements (Andreadis et al., 1999). Despite all the advantages of retroviral vectors there are some problems: low transduction efficiencies; cell division is required for integration into the genome of the target cell; low viral titters production; and low stability (Andreadis et al., 1999; Cruz et al., 2000). ∗ Corresponding author. Tel.: +351-21-442-77-87; fax: +351-21-442-11-61. E-mail address: [email protected] (M.J.T. Carrondo).
0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.03.007
Several methods can be used to quantify retroviral vectors: titration assays (Forestell et al., 1995; Lee et al., 1996) and reverse transcriptase activity assays (Kwon et al., 2003) to quantify infectious particles; and electron microscopy to quantify total particles (Higashikawa and Chang, 2001). Other methods exist that are semi-quantitative such as RT-PCR (Heinemeyer et al., 1997) and immunoblotting (Muriaux et al., 2002). Some new methods have been developed from the conventional PCR method such as real-time PCR that with on-line and real-time monitoring of fluorescent-labelled PCR products enables a rapid and quantitative determination of sample DNA concentration, without the need of gel products detection (Rasmussen, 2001; Müller and Wirth, 2002). This quantitation is achieved using software that calculates the copy number of the templates in respect to a standard curve generated in parallel. Slopes of the fluorescence accumulation curves are calculated during the log-linear phase of amplification so that the quantitation is based on kinetic analyses not relying
116
M. Carmo et al. / Journal of Virological Methods 119 (2004) 115–119
on end-point PCR products (Rasmussen, 2001; Saha et al., 2001). In order to reduce the time needed for MLV-based vector quantitation, a real-time RT-PCR method was developed. This method quantifies vectors without an RNA extraction step using avian myeloblastosis virus (AMV) reverse transcriptase and LightCycler technology.
2. Materials and methods 2.1. Production of retroviral vectors Retroviral vectors were obtained from supernatant of human TE FLY A7 packaging cell line, derived from TE 671 cells (ECACC no. 89071904) transformed with the plasmid pMFGSnlsLacZ (Pizzato et al., 2001) that produces murine leukaemia virus-based vectors; this cellular line was kindly provided by Dr. Otto Merten from Généthon, France. These cells were cultured in DMEM medium (Gibco, Paisley, UK) supplemented to have 4.5 g/L of glucose (Merck, Darmstadt, Germany), 6 mM of glutamine (Gibco) and 5% FBS (Gibco). After three days of culture the medium was replaced and vectors were produced during 24 h. 2.2. Choice of standard and primers The plasmid pSIR (Clontech, Palo Alto, USA) was chosen as standard to quantify retroviral RNA; pSIR is a plasmid derived from Moloney murine leukaemia virus. Forward and reverse primer sequences specific for MLV were located within the LTR region and consisted of 5 -ATT GAC TGA GTC GCC CGG-3 and 5 -AGC GAG ACC ACA AGT CGG AT-3 , respectively. 2.3. Pre-treatment of supernatants Cell culture supernatants were filtered with 0.45 m pore filters to remove cells and cellular debris and incubated at 75 ◦ C for 10 min to release RNA. In order to destroy DNA derived from lysed cells 2 l DNaseI (1 U/l, Sigma, Steinheim, Germany) and 4 l 25mM MgCl2 were incubated with 100 l of filtered supernatant for 30 min at room temperature. 2.4. cDNA synthesis cDNA synthesis was performed using first strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). DNase I pre-treated supernatants were incubated for 10 min at 75 ◦ C. Following this, 11.5 l of cDNA synthesis mix was prepared to the indicated end-concentrations: 2 l of reaction buffer (1×), 4 l MgCl2 (5 mM), 2 l dNTP (1 mM), 1.7 reverse primer (1 M), 1 l RNase inhibitor (50 U) and 0.8 l AMV reverse transcriptase (20 U). The
8.5 l of supernatant was added to the cDNA synthesis mix, incubated for 10 min at 25 ◦ C and then incubated for 1 h at 42 ◦ C. A subsequent heat inactivation step of 5 min at 99 ◦ C was performed and the final cDNA was stored at −20 ◦ C. 2.5. Real-time PCR For LightCycler reaction a mastermix of the following reaction components was prepared to the indicated end-concentrations: 2 l LightCycler master (Fast start DNA master SYBR Green I; Roche Diagnostics), 3.2 l MgCl2 (4 mM), 0.6 l forward primer (0.5 M), 0.86 l reverse primer (0.5 M) and 3.34 l PCR grade water. LightCycler master mix was distributed into the LightCycler capillaries (10 l each) and 10 l of cDNA or pSIR standard were added as PCR template. Capillaries were closed and placed into the light cycler rotor. The following LightCycler run protocol was used: denaturation program (95 ◦ C for 10 min); amplification and quantitation program repeated 45 times (60 ◦ C for 10 min; 72 ◦ C for 10 min with a single fluorescent measurement); melting curve program (65–95 ◦ C for 10 min with continuous fluorescent measurement); and finally a cooling step to 40 ◦ C. For visualisation, 10 l of the reaction products were separated by gel electrophoresis on 2% agarose gel in TBE buffer containing ethidium bromide. In case of successful amplification a product around 73 bp in length indicated the presence of murine C-type retroviral nucleic acids. 2.6. Quantitation of infectious particles For determination of the concentration of retroviral infectious particles, target cells HCT 116 (ATCC no. CCL-247) were seeded in 96-well, flat-bottomed plates (Starstedt, Newton, USA) at a density of 5 × 104 cells/cm2 and incubated during 24 h. Infections were then carried out by replacing the medium with 20 l of dilutions (10−1 to 10−4 ) of viral supernatants in DMEM medium containing 8 g/ml of polybrene (Sigma) followed by incubation at 37 ◦ C for 4 h. After this time 180 l of fresh medium was added to the plate, and infected cells were incubated for 1 day. Then, the medium of each well was aspirated and washed with 100 l of PBS. The 100 l of fixing solution, with 0.75% formaldehyde 37% (Merck) and 5.1% of glutaraldehyde (Sigma) in PBS, was added to each well and left for 2 min before aspiration; then each well was washed with 100 l of PBS. Next, 100 l of the dye consisting of 5 mM K3 Fe8 (CN)6 (Merck), 5 mM K4 Fe(CN)6 (Merck), 1 mM MgCl2 (Merck) and 200 mg/ml X-gal (Stratagene, La Jolla, USA) in DMF (Riedel deHaën, Seelze, Germany) was added to each well, and the LacZ-positive (blue) cells were counted after 24 h of incubation at 37 ◦ C. For each sample three dilution sets were performed.
M. Carmo et al. / Journal of Virological Methods 119 (2004) 115–119
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3. Results
Table 1 Method efficiencya
3.1. Standard curve validation
Slopestandard
Estandard
Slopesamples
Esamples
Estandard − Esamples
−3.38 −3.44 −3.42
1.98 1.95 1.96
−3.32 −3.39 −3.32
2.00 1.97 2.00
0.03 0.02 0.04
In LightCycler real-time RT-PCR a standard curve must be used to quantify DNA. A DNA plasmid (pSIR) with MLV sequence was chosen as standard in this method. After a few optimisations (plasmid purification by precipitation with ammonium acetate and quantitation using GeneQuantTM , Amersham, UK) a standard curve with good efficiency and reproducibility was obtained. The efficiency is shown by the slope of the standard curve with the log of the initial template copy number plotted horizontally and the cycle number at the crossing point plotted vertically. In this case the slope obtained was around 3.4 (must be between 3.3 and 3.9), the reproducibility was seen calculating the variation coefficient between three repetitions in a row and this was less than 1%. 3.2. Confirmation of primer specificity Specificity of real-time RT-PCR products was verified by agarose gel electrophoresis (2%) and resulted in a single product around 70 bp, as desired (Fig. 1). In addition a LightCycler melting curve was performed which resulted in single product with specific melting temperature of 85 ◦ C. 3.3. RNA quantitation After standard curve optimisation and primer specificity confirmation a test must be done to evaluate the method efficiency amplifying retroviral vector RNA. In order to have high method efficiency, the difference between efficiency of the standard curve and efficiency of the RNA sample curve must be lower than 0.05 and the amount of PCR product present at the crossing point must be the same for standards and samples (Rasmussen, 2001), with the efficiency being calculated as E = 10(−1/Slope) . The test was repeated three
a Comparison between efficiency of the standard curve (E standard ) and efficiency of an RNA samples curve (Esamples ).
Table 2 Intra- and inter-assay variability, standard deviation and coefficient of variability (CV) for three experiments in a row Average RNA copy (l)
S.D.
CV intra-assay (%)
CV inter-assay (%)
4.33 × 103 5.31 × 103 4.50 × 103
8.30 × 101 6.66 × 101 1.09 × 102
2 1 2
3
times in a row and the difference of efficiency was always lower than 0.05 and the amount of PCR product present at the crossing point was the same for standards and samples. These results are shown in Table 1. 3.4. Intra- and inter-assays To confirm the accuracy and reproducibility of real-time RT-PCR the intra-assay variation was determined in three repeats within a LightCycler run. Inter-assay variation was investigated in three different experimental runs performed on 3 days using three different master mixes of LightCycler Fast start DNA master SYBR green I kit (Roche diagnostics). Test variation was 2% in intra-test experiments and 3% in inter-test experiments, as shown in Table 2. 3.5. Sensitivity For evaluation of assay sensitivity serial dilutions of supernatants with retroviral vectors were subjected to real-time RT-PCR. The results show that with this method retroviral vectors can be amplified from a minimum concentration of 3 × 102 RNA copies/l. 3.6. RNA concentration versus infectious vectors To understand the relationship between vectors with RNA and infectious vectors, three samples of vectors were quantified by real-time RT-PCR and titre assay in the same day. The results are presented in Table 3. It can be noticed that the Table 3 Comparison between vectors with RNA (RNA V) and infectious vectors (IV)
Fig. 1. Specificity of real-time RT-PCR. M: PCR markers (Promega, Madison, USA) 1000, 750, 500, 300, 150, 50 bp; lanes 1–6: pSIR plasmid; lane 7: H2 O control; lane 8–11: supernatant sample.
RNA V
IV
RNA V/IV
1.38 × 109 1.80 × 109 1.73 × 109
1.52 × 107 1.95 × 107 2.00 × 107
91 92 89
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ratio of vectors with RNA and infectious vectors is around 90 with a coefficient of variability of 2%, meaning that the majority of the vectors that contain RNA are not infectious.
4. Discussion We describe a real-time RT-PCR method for quantitation of retroviral vector particles with RNA. The presented protocol can be used for any MLV-based retroviral vectors since the quantitation is performed with primers specific for the LTR region. Real-time RT-PCR is a highly sensitive method that allows quantitation of rare transcripts. It is also easy to perform, provides necessary accuracy and produces reliable and rapid quantitation results (Pfaffl, 2001). With this method two quantitation types are possible: a relative quantitation based on the relative expression of a target gene versus a reference gene; and an absolute quantitation based either on an internal or an external calibration curve (Rasmussen, 2001). The method uses plasmid DNA as standard; this has the advantage of being a standard easy to quantify and stable. Some methods exist that use real-time PCR to quantify retroviral vector particles with culture supernatant containing retroviral particles as standard. But retroviral vectors are very unstable having a half-life of 5–8 h at 37 ◦ C (Andreadis et al., 1999; Higashikawa and Chang, 2001; McTaggart and Al-Rubeai, 2002) and are sensitive to freezing and thawing, making reproducibility very difficult (Lee et al., 1996; Higashikawa and Chang, 2001). Another problem with these methods is that the standard supernatants are monitored overtime by their infectious particle titre, not by their RNA copy content, since this is used as standard. Another advantage of the new method is that it does not need an RNA extraction step. A heat step at 75 ◦ C is used to destroy vector particles, RNA becoming free in solution. Furthermore, this method presents low variability between the intra-assay tests (2%), ensuring a good precision, and low variability between the inter-assay tests (3%) meaning that the method has good reproducibility. Normally, to quantify infectious retroviral vectors a titre assay is used that take 5 days for completion (Forestell et al., 1995; Lee et al., 1996), whereas the method presented here has the ability to quantify vectors in a few hours. However, the production of defective particles is a problem concerning retroviral vectors production (Forestell et al., 1995; Higashikawa and Chang, 2001). These particles can be produced without envelope, without RNA or with RNA but not being infectious. The method described quantifies all vector particles containing RNA. The establishment of a correlation between vectors with RNA and infectious vectors for each specific situation can solve this problem, since this ratio can be applied to quantitation. A test was made to calculate the ratio between vectors with RNA and infectious particles; this ratio was found to be around 90 with a variability of 2%, for the standard production conditions defined in this work. So to quantify infectious vectors through this method
the RNA copy number obtained must be divided by this ratio. The new method is also useful to study vector stability by comparison of infectious vectors with vectors with RNA and total vectors. The smaller the differences between these three types of vectors the more stable are the vectors produced. With minor modifications, this method also can be used to study other retrovirus, even true RNA viral families. In conclusion, the retroviral vector quantitation method described above shows a very good reproducibility in interand intra-assays. By using a plasmid as a standard, it is ensured that the results are consistent between different production runs. The sensitivity of the method is low enough to allow the characterisation of most culture supernatants. Moreover, this method can be used for the determination of infectious particles under standard production conditions, thus allowing a faster quantitation of the vectors produced. Finally, it is possible, by including this method in a broader vector analysis strategy, to have a better characterisation of the produced supernatant and to study the effects of vector inactivation during downstream processing and storage as well as in the quality analysis of clinical-grade material.
Acknowledgements The authors acknowledge the financial support received from the European commission (QLK3-CT-2002-01949) and the Fundação para a Ciˆencia e Tecnologia, Portugal (POCTI SFRHBD31282000 and POCTI/1999/BIO/35695).
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