Optimized Reagents for Probe-Based qPCR using the GoTaq® Probe qPCR and RT-qPCR Systems
Promega Corporation
Publication Date: January 2014; tpub 115
Abstract
Amplification using quantitative PCR (qPCR) is measured by detecting changes in a fluorescent signal. Fluorescently labeled probes are frequently used as the fluorescent reporter in qPCR. The GoTaq® Probe qPCR and RT-qPCR Systems are ready-to-use 2X master mixes optimized for probe-based qPCR detection chemistries. The GoTaq® Probe Master Mixes are designed to provide resistance to a wide range of PCR inhibitors. The master mixes also employ rapid hot-start activation and processive enzymes, making them compatible with both standard and FAST instrument cycling programs. The 1-step and 2-step RT-qPCR systems also include GoScript™ Reverse Transcriptase, to enable efficient synthesis of first-strand cDNA in preparation for PCR amplification.
Introduction
Since its inception, real-time PCR, known as qPCR, has become a standard tool in the analysis of nucleic acids. By monitoring a change in a fluorescent reporter signal, qPCR methods detect and measure the accumulation of the product as the PCR amplification proceeds. This allows a high level of sensitivity and a wide dynamic range, resulting in accurate quantitation (1) .
Popular qPCR detection methods include probe-based as well as dye-based detection chemistries. Probe-based qPCR uses a fluorescent primer or probe to detect the amplification product. An advantage to using a labeled primer or probe is that it allows sequence-specific detection of the target, which reduces the likelihood of detecting nonspecific artifacts. Probe-based detection also can accommodate multiplex amplification, through the use of differentially labeled probes. There are a number of different approaches to probe-based qPCR detection, including hydrolysis probes, hybridization probes, hairpin probes and labeled primers (2) .
The GoTaq® Probe qPCR and RT-qPCR Systems are optimized for qPCR assays using probe-based detection chemistries; experiments performed with the GoTaq® Probe qPCR Master Mix exhibit sensitive quantification and robust performance.
Methods
2-Step RT-qPCR: In preparation for 2-step RT-qPCR, first-strand cDNA was synthesized from RNA templates using the GoScript™ Reverse Transcription System. Following heat-inactivation of the reverse transcriptase, the cDNA was used as the template in 2-step RT-qPCR amplification reactions with the GoTaq® Probe qPCR Master Mix.
qPCR Amplification Conditions: Unless otherwise noted, 20μl qPCR and RT-qPCR reactions were prepared. The GoTaq® amplification reactions included carboxy-X-rhodamine (CXR) as a passive reference dye. The GoTaq® Probe Master Mix formulations do not contain a reference dye; however, a separate tube of CXR reference dye is included with the system, allowing users to add reference dye if desired. Addition of the reference dye helps maximize effectiveness of the GoTaq® Probe qPCR and RT-qPCR Systems when used on real-time PCR instruments that allow for normalization.
qPCR Amplification Conditions: Tables 1, 2 and 3 show the cycling conditions used in these experiments.
Results
High Sensitivity and Wide Dynamic Range with Hydrolysis Probes. One of the most commonly used label-based detection methods is the hydrolysis probe chemistry. This chemistry uses a labeled probe designed to anneal to the sequence between two unlabeled amplification primers. Attached to the 5´end of this probe is a fluorescent reporter, and on the 3´end is a quencher. In solution, the fluorescent reporter and the quencher molecule are in close proximity, which results in quenching of the fluorescent dye. During amplification, the 5´ exonuclease activity of the Taq polymerase degrades the probe. Once the probe is degraded, the reporter and quencher molecules are released, resulting in an increase in fluorescence; the amount of fluorescence generated is proportional to the amount of product produced (3) . When compared to a popular hydrolysis probe system (Vendor L), the GoTaq® Probe qPCR and RT-qPCR Systems performed comparably, demonstrating efficient amplification with hydrolysis probe qPCR assays (Figures 1 and 2).
Scalable Reaction Volume. The GoTaq® Probe qPCR Systems can accommodate a range of reaction volumes from 5µl to 50µl with no change in performance (Figure 3). This scalability gives the user greater flexibility in choosing the instrument, throughput and plate format.
Robust Amplification with Hairpin Probes. Hairpin probe assays use a labeled probe designed to form a hairpin structure in solution; a fluorescent reporter is covalently attached to one end of this probe, and on the other end of the probe is a quencher. When the probe is in a hairpin conformation, the reporter and quencher are in close proximity, and fluorescence is quenched. During amplification, the probe favors hybridization to the target sequence; when the hairpin dissociates, the reporter and quencher molecules are no longer held in close proximity, resulting in an increase in fluorescence (4) . Using a hairpin probe (Molecular Beacons; Figure 4), the GoTaq® Probe qPCR Systems demonstrated robust performance.
Resistance to Inhibitors. A wide variety of sample contaminants can inhibit qPCR assays. Potential inhibitors include endogenous cellular contaminants, such as nucleases and other proteins, carbohydrates, or (in the case of RNA samples) genomic DNA contamination. In addition, nucleic acids isolated from environmental samples are notorious for carrying over inhibitors, including hematin from blood, humic acid from soil, dyes from textiles, and bile or urea from fecal or urine samples. Depending on the purification method used, it is possible to introduce inhibitors during the nucleic acid extraction process, such as phenol, guanidine, alcohols or detergents (5) . As shown in Figure 5, the GoTaq® Probe qPCR Master Mix demonstrates resistance to a wide range of PCR inhibitors.
Duplex Amplification. Probe-based detection chemistries give users the potential to perform multiplex amplifications using differentially labeled probes. A multiplexed approach can increase throughput by allowing users to amplify multiple targets per well. For example, amplifying a gene of interest and reference transcript in the same well using the same template can reduce variability in relative quantitation assays (6) . Multiplexing also conserves master mix reagents, as well as precious sample material. The GoTaq® Probe qPCR Master Mix demonstrated good amplification efficiency both as monoplex and duplexed reactions (Figure 6).
Standard and FAST Cycling. FAST cycling can be another approach to increase qPCR throughput. FAST cycling time is determined by several factors including: the activation time required by the hot-start Taq DNApolymerase, the thermal cycling times (more processive enzymes can better accommodate faster cycling) and the ramp rates, which are dependent on the instrument hardware.
The GoTaq® Probe qPCR Master Mixes can accommodate either standard- or FAST-cycling conditions (Table 4). The GoTaq® activation step can be as short as 20 seconds, although a 2-minute initial denaturation can be helpful if the sample template is high molecular weight genomic DNA.
Summary
The GoTaq® Probe qPCR and RT-qPCR Systems demonstrate sensitive detection and quantification over a wide range of DNA or RNA targets when used with probe-based detection assays. The master mixes also provide resistance to a wide range of PCR inhibitors, helping to ensure robust amplification. Rapid hot-start activation and processive enzymes allow the GoTaq® Probe Systems to support both standard-and fast-cycling methods, and the GoTaq® Probe qPCR Master Mix demonstrates efficient amplification of both monoplex and duplexed reactions.
Related Resources
Article References
- Valasek, M.A. and Repa, J.J. (2005) The Power of Real-Time PCR Adv. Physiol. Educ. 29, 151–9.
- VanGuilder, H.D., Vrana, K.E. and Freeman, W.M. (2008) Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques 44, 619–26.
- Heid, C.A. et al. (1996) Real time quantitative PCR Genome Res. 6, 986–94.
- Tyagi, S. and Kramer F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303.
- Bessetti, J. (2007) An Introduction to PCR Inhibitors Profiles in DNA 10, 9–10.
- Bustin, S.A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): Trends and problems. J. Mol. Endocrinol. 29, 23–39.
How to Cite This Article
Scientific Style and Format, 7th edition, 2006
Schmidt, B. and Nassif, N. Optimized Reagents for Probe-Based qPCR using the GoTaq® Probe qPCR and RT-qPCR Systems. [Internet] January 2014; tpub 115. [cited: year, month, date]. Available from: https://www.promega.com/resources/pubhub/optimized-reagents-for-probe-based-qpcr-using-the-gotaq-probe-qpcr-and-rt-qpcr-systems/
American Medical Association, Manual of Style, 10th edition, 2007
Schmidt, B. and Nassif, N. Optimized Reagents for Probe-Based qPCR using the GoTaq® Probe qPCR and RT-qPCR Systems. Promega Corporation Web site. https://www.promega.com/resources/pubhub/optimized-reagents-for-probe-based-qpcr-using-the-gotaq-probe-qpcr-and-rt-qpcr-systems/ Updated January 2014; tpub 115. Accessed Month Day, Year.
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