Our results at Wyeth Research show that QuantiTect®. Multiplex Kits from QIAGEN are well suited for multiplex analysis of up to four RNA targets in two-step and ...
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Application Forum
Simultaneous Quantification of Four RNA Targets by Multiplex, Real-Time RT-PCR without Optimization John W. Coleman, J. Erik Johnson, and David K. Clarke Wyeth Vaccines Research, NY, USA
INTRODUCTION Researchers carrying out quantitative, multiplex, realtime PCR and RT-PCR require master mixes that provide accurate, simultaneous quantification of multiple targets in the same reaction without time-consuming optimization. Our results at Wyeth Research show that QuantiTect® Multiplex Kits from QIAGEN are well suited for multiplex analysis of up to four RNA targets in two-step and onestep real-time RT-PCR, requiring no PCR optimization and providing the same efficiency as single-target amplification (singleplex) reactions. Multiplex, real-time PCR refers to the use of multiple fluorescently-labeled oligonucleotide probes for the quantification of amplicons produced by different primer pairs in the same reaction. This method is becoming more common with the introduction of cyclers capable of spectrally resolving different fluorophores (e.g., Applied Biosystems® 7500, Mx3000P®, LightCycler® 480, and iCycler iQ®), and with the development of master mixes that facilitate the quantification of multiple targets in the same reaction tube. Some master mixes intended for singleplex analysis have been adapted for multiplex analysis1, 2, but require time-consuming optimization of primer–probe sets. Therefore, evaluating and selecting a suitable master mix is critical when establishing multiplex assays. APPLICATION OF MULTIPLEX, REAL-TIME PCR TO VACCINE RESEARCH Multiplex analysis can be applied to many different fields, such as vaccine research. To advance studies of new vaccine vectors, we are developing sensitive real-time RT-PCR assays to monitor the outcome of vaccination of animals3. An important aspect of characterizing new viral vectors is to analyze their distribution and persistence in small animal models. For vesicular stomatitis virus (VSV) vector development, a sensitive method to detect vector nucleic acid and a vector-encoded gene (i.e., viral RNA targets) is required in order to monitor the extent and duration Vol. 43 ı No. 4 ı 2007
of vector replication. Since we are interested in quantifying these viral RNA targets as well as an animal housekeeping gene (to normalize viral RNA levels) and exogenous RNA (to monitor RNA purification efficiency), we investigated the feasibility of 4plex, real-time RT-PCR. Analyzing targets in the same well, instead of in separate wells, would avoid potential problems with well-to-well variation in starting template amount and reaction conditions. OPTIMIZED MASTER MIXES FOR MULTIPLEX, REAL-TIME PCR QIAGEN offers two kits for multiplex analysis: the QuantiTect Multiplex PCR Kit (for PCR and two-step RT-PCR procedures) and the QuantiTect Multiplex RT-PCR Kit (for one-step RT-PCR procedures). The optimized PCR master mix in the kits ensures that amplicons are amplified with equal efficiency and sensitivity in both multiplex and singleplex reactions. This is achieved through various reaction components in the master mix that create the conditions for high specificity in PCR. Firstly, a balanced combination of K+ and NH4+ ions and a novel PCR additive called synthetic factor MP work together to promote stable and efficient annealing of primers to the nucleic acid template. Secondly, a stringent hot-start Taq DNA polymerase becomes active only after incubation at 95°C, preventing the formation of nonspecific products and primer–dimers in every PCR cycle. The QuantiTect Multiplex RT-PCR Kit is also supplied with an RT mix for highly efficient and sensitive reverse transcription over a wide range of RNA template amounts. EVALUATION OF QUANTITECT MULTIPLEX KITS To determine whether the QuantiTect Multiplex RT-PCR Kit could amplify four targets simultaneously in multiplex, real-time, one-step RT-PCR, we analyzed RNA standards (synthetic oligonucleotides) or RNA purified from nasal turbinate homogenates from mice inoculated intranasally with a VSV vector encoding an HIV-1 gene. The four targets were the viral nucleocapsid gene (N), the HIV-1 gene, gag (G), a www.biotechniques.com ı BioTechniques ı 517
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mouse housekeeping gene, RPLO (R), and a spiked exogenous control, Armored-RNA (A). We also analyzed these four targets by multiplex, real-time, two-step RT-PCR using the QuantiTect Multiplex PCR Kit. The sensitivity and specificity of the 4plex assays were determined by comparison with the corresponding singleplex assays.
(Fig. 1D). The sensitivity of the 4plex and singleplex assays was comparable, with detection of 10–100 copies for each gene. This sensitivity was maintained when mixed targets were done with genes N, R and A fixed at 105 copies and gene G fixed at 100 copies or genes G, R and A fixed at 105 copies and gene N fixed at 100 copies (data not shown). The specificity of the four primer–probe sets was confirmed in control reactions in which the template molecules were absent/modified (data not shown).
MULTIPLEX, REAL-TIME, ONE-STEP RT-PCR USING STANDARDS Multiplex, real-time, one-step RT-PCR was carried out using 10-fold serial dilutions (from 107 to 101 copies) of synthetic RNA oligonucleotides of three of the amplicons (N, G, R) and 105 copies of the spiked control (A). Reaction setup and cycling were carried out according to the QIAGEN protocol for 4plex analysis with the QuantiTect Multiplex RT-PCR Kit on the Applied Biosystems 7500. Probes were labeled with 6-FAM (N gene), VIC® (G gene), NED (R gene), and Cy®5 (A gene), and primers and probes were each used at 0.2 μM. For the singleplex reactions, the probe concentration was set at 0.2 μM, while the primer concentrations were increased to 0.4 μM. The amplification plots achieved with the 4plex assay overlapped with those from the singleplex assays, indicating equivalent threshold cycle numbers (CT) at each dilution for gene N (Fig. 1A), gene G (Fig. 1B), gene R (Fig. 1C), and gene A
A
Viral/mouse RNA purified from nasal turbinate homogenates was analyzed by real-time, one-step RT-PCR in both 4plex and singleplex assays. Reactions were run using the QuantiTect Multiplex RT-PCR Kit and the recommended primer–probe concentrations and cycling conditions. A comparison of the 4plex and singleplex assays showed equivalent CT values for each gene (Table 1). MULTIPLEX, REAL-TIME, TWO-STEP RT-PCR USING VIRAL/MOUSE RNA To determine whether the QuantiTect Multiplex PCR Kit could be used in real-time, two-step RT-PCR, viral/mouse
B
N-FAM 107
106
105
104 103 102
Assay Singleplex 4plex
C
MULTIPLEX, REAL-TIME, ONE-STEP RT-PCR USING VIRAL/MOUSE RNA
107
101
Slope -3.36 -3.36
104
103 102
Assay Singleplex 4plex
104
103 102
101
Slope -3.50 -3.51
R2 0.9998 0.9988
A-Cy5 105
101
Slope -3.30 -3.30
105
Assay Singleplex 4plex
D 105
106
R2 0.9995 0.9998
R-NED 107 106
G-VIC
R2 0.9974 0.9969
Figure 1. Comparison of amplification plots for detection of genes N, G, R, and A in a 4plex assay and corresponding singleplex assays using the QuantiTect Multiplex RT-PCR Kit and four different fluorescently-labeled probes. The templates were 10-fold serial dilutions (from 107 to 101 copies) of synthetic RNA oligonucleotides of gene N (A), gene G (B), and gene R (C), and 105 copies of gene A (D). Down to 10 copies of genes N, G, and R were detected.
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Vol. 43 ı No. 4 ı 2007
Table 1 CT values for the quantification of four RNA targets by real-time, one-step RT-PCR in 4plex and singleplex assays using the QuantiTect Multiplex RT-PCR Kit CT VALUESA FOR MOUSE NASAL TURBINATESB GENE (LABEL) N (FAM) G (VIC) R (NED) A (Cy5)
ASSAYC,D
SAMPLE 1
SAMPLE 2
SAMPLE 3
SAMPLE 4
SAMPLE 5
4plex
21.67
23.27
21.09
21.85
22.75
Singleplex
21.03
22.99
20.35
20.98
21.74
4plex
24.37
25.07
23.26
24.34
24.61
Singleplex
24.78
25.12
23.73
24.65
25.51
4plex
22.05
21.81
21.70
22.50
21.92
Singleplex
21.65
21.47
21.16
21.99
21.30
4plex
26.49
26.22
25.75
27.51
27.19
Singleplex
27.41
27.17
26.55
28.48
27.52
Table 2 CT values for the quantification of four RNA targets by real-time, two-step RT-PCR in 4plex and singleplex assays using the QuantiTect Multiplex PCR Kit CT VALUESA FOR MOUSE NASAL TURBINATESB GENE (LABEL) N (FAM) G (VIC) R (NED) A (Cy5)
ASSAYC,D
SAMPLE 1
SAMPLE 2
SAMPLE 3
SAMPLE 4
SAMPLE 5
4plex
26.48
29.06
26.36
26.58
26.59
Singleplex
27.00
29.99
26.80
27.08
27.05
4plex
25.45
27.80
25.89
25.32
25.62
Singleplex
25.81
28.13
26.26
25.86
25.92
4plex
22.44
24.38
22.85
21.80
23.27
Singleplex
22.32
24.31
22.60
21.62
22.90
4plex
26.16
26.47
26.11
25.29
25.60
Singleplex
26.76
27.34
26.66
26.04
26.18
CT is the threshold cycle number at which fluorescence was detected above the background. Positive CT values are ≤36, while negative CT values are >36. B Nasal turbinate tissues are from mice which were individually and intranasally inoculated. C Separate singleplex reactions for genes N, G, R, and A were carried out in different wells containing primer–probe sets that were specific for each gene. D The 4plex reaction was carried out in one well containing primer–probe sets for genes N, G, R, and A. A
RNA from nasal turbinate homogenates was analyzed in 4plex and singleplex assays. Equivalent CT values were achieved for each gene regardless of whether a 4plex or a singleplex assay was performed (Table 2). CONCLUSION QuantiTect Multiplex Kits accurately quantified multiple targets simultaneously in the same reaction tube when tested with RNA standards or with total/viral RNA from mouse tissues. This helped us at Wyeth Research to increase the throughput of our animal studies and to reduce reagent costs and concerns about false-negatives. Since our target genes may be expressed at different levels after animal inoculation, lengthy optimization of primer–probe sets and limiting primer experiments were often necessary with other master mixes. These steps were necessary in order to prevent abundant genes from dominating the amplification reaction and affecting detection of less abundant genes. With QuantiTect Multiplex Kits, this time-consuming optimization was not needed. Whether these kits were used Vol. 43 ı No. 4 ı 2007
in two-step or one-step RT-PCR, they provided sensitive and specific detection of target genes, giving equivalent CT values in both 4plex and singleplex assays. Since optimization of primer–probe sets and cycling conditions are not necessary, QuantiTect Multiplex Kits represent an advanced tool for quantitative, real-time PCR and RT-PCR. REFERENCES 1.Johnson, B.W. et al. J. Clin. Microbiol. 43, 4977–4983 (2005). 2.Scheltinga, S.A. et al. J. Clin. Virol. 33, 306–311 (2005). 3.Coleman, J.W. et al. J. Virol. Meth. 143, 55-64 (2007).
TRADEMARKS Trademarks: QuantiTect® (QIAGEN Group); Applied Biosystems®, VIC® (Applera Corporation or its subsidiaries); Cy® (GE Healthcare); iCycler iQ® (Bio-Rad Laboratories, Inc.); LightCycler® (Roche Group); Mx3000P® (Stratagene).
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