Optimized Reagents for Probe-Based qpcr using the GoTaq Probe qpcr and RT-qPCR Systems 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. Ben Schmidt and Nadine Nassif Promega Corporation Publication Date: January 2014; tpub 115 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. 1 Schmidt, B. and Nassif, N.
qpcr Amplification Conditions: Tables 1, 2 and 3 show the cycling conditions used in these experiments. Table 1. Standard cycling for qpcr and 2-step RT-qPCR with the GoTaq Probe Systems. Table 2. Standard cycling for 1-step RT-qPCR with the GoTaq Probe Systems. Table 3. FAST cycling for qpcr and 2-step RT-qPCR with the GoTaq Probe Systems. 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). 2 Schmidt, B. and Nassif, N.
Figure 1. GoTaq Probe 2-Step RT-qPCR System with dutp. cdna was generated from Human Control RNA (Applied Biosystems; Cat.# 4307281), using the GoScript Reverse Transcription System. Amplification (standard cycling) was performed using a human GAPDH TaqMan assay (Applied Biosystems; Cat.# Hs02758991_g1). The human GAPDH sequence was detected from ten-fold serial dilutions of cdna (100ng to 1pg), using GoTaq Probe qpcr Master Mix for amplification. Panel A. Standard curve. Panel B. The GoTaq Probe 2-Step RT-qPCR System performance was compared to that of the TaqMan Universal Master Mix (Applied Biosystems; Vendor #4). 3 Schmidt, B. and Nassif, N.
Figure 2. GoTaq Probe 1-Step RT-qPCR System with dutp. Human Control RNA (Applied Biosystems: 4307281) was serially diluted ten-fold and added to the GoTaq Probe 1-Step RT-qPCR System reactions (100ng to 0.1pg per reaction). Onestep amplification (standard cycling) was performed using a human GAPDH TaqMan assay (Applied Biosystems: Hs02758991_g1). The human GAPDH sequence was detected using the GoScript RT Mix for 1-Step RT-qPCR plus the GoTaq Probe Master Mix with dutp for 1-step amplification; the resulting standard curve is shown (Panel A). Equivalent performance was observed when comparing the GoTaq Probe 1-Step RT-qPCR System with dutp to the TaqMan RNA-to- Ct 1-Step Kit from Applied Biosystems (Vendor #4) (Panel B). 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. 4 Schmidt, B. and Nassif, N.
Figure 3. Scaling reaction volumes between 50µl and 5µl of target RNA with the GoTaq Probe 2-Step System. cdna was generated from human heart total RNA (BioChain: R1234122-50), using the GoScript Reverse Transcription System. Amplification (standard cycling) was performed using a human GAPDH TaqMan assay (Applied Biosystems: Hs02758991_g1). Reaction volumes of 50μl were amplified in 96-well plates using an Applied Biosystems 7500 instrument (Panel A). Reaction volumes of 5μl were amplified in 384-well plates using a Roche LightCycler 480 instrument (Panel B). The human GAPDH sequence was detected from five-fold serial dilutions of cdna (100ng to 6.4pg), using GoTaq Probe qpcr Master Mix for amplification. The resulting standard curves are shown. 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. 5 Schmidt, B. and Nassif, N.
Figure 4. Molecular Beacons Amplification using the GoTaq Probe 2-Step RT-qPCR System. cdna was generated from human heart total RNA (BioChain: R1234122-50), using the GoScript Reverse Transcription System. Amplification (standard cycling) was performed using Molecular Beacon probe and primer sequences for the human GAPDH gene (obtained from Sigma- Proligo design and synthesis services). Final (1X) primer and probe concentrations used in amplification were 1µM and 0.34µM, respectively. The human GAPDH sequence was detected from five-fold serial dilutions of cdna (100ng to 6.4pg), using GoTaq Probe qpcr Master Mix for amplification; the resulting standard curves are shown. 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. 6 Schmidt, B. and Nassif, N.
Figure 5. GoTaq Probe qpcr Systems are perform well in the presence of common PCR inhibitors hematin, humic Acid and phenol cdna was generated from human liver total RNA (BioChain R1234149-50), using the GoScript Reverse Transcription System. Amplification (standard cycling) was performed using the GoTaq Probe qpcr Master Mix, detecting 10ng of cdna template with a human GAPDH TaqMan assay (Applied Biosystems Hs02758991_g1). Panel A. The effect of hematin on the performance of the GoTaq Probe qpcr Master Mix in comparison to the itaq Universal Probes Master Mix (BioRad; Vendor #1) was evaluated by adding hematin to amplification reactions at increasing concentrations; final concentrations of 0, 15, 20, 25, 30, and 50µM were added to the reactions. The GoTaq Probe qpcr master mix remains unaffected by the increasing hematin concentration, while the itaq Universal Probes Master Mix shows no amplification with any amount of the inhibitor present. Panel B The effect of humic acid on the performance of the GoTaq Probe qpcr Master Mix in comparison to the QuantiTect Probe Master Mix (Qiagen; Vendor #2) was evaluated by adding increasing amounts humic acid to amplification reactions (0, 200, 300, 400, 600, and 800ng). The GoTaq Probe qpcr master mix begins to show a decreased change in fluorescence at 400ng of inhibitor. In comparison, the QuantiTect Probe Master Mix demonstrates a shift in Cq and a decreased change in fluorescence at just 200ng of inhibitor; at 400ng and above, no amplification is observed. Panel C. The effect of phenol on the performance of the GoTaq Probe qpcr Master Mix in comparison to the Maxima Probe Master Mix (Thermo; Vendor #3) was evaluated by adding increasing amounts phenol to amplification reactions (0, 0.25, 0.5, 1, 1.5 and 2%). As phenol is increased to 1.5%, the GoTaq Probe qpcr master mix shows a gradual decrease in the change in fluorescence, although the Cq remains unchanged. In comparison, the Maxima Probe Master Mix demonstrates a dramatic reduction in change of fluorescence at 1% phenol, and no amplification at higher amounts. 7 Schmidt, B. and Nassif, N.
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). Figure 6. Multiplex Amplification with the GoTaq Probe 2-Step RT-qPCR System. cdna was generated from human heart total RNA (BioChain R1234122-50), using the GoScript Reverse Transcription System. Amplification (standard cycling) was performed using a human GAPDH TaqMan assay (JOE probe; Applied Biosystems 402869) as well as a human TNNT2 TaqMan assay (FAM probe; Applied Biosystems Hs00165960_m1). Standard curves were generated with each TaqMan assay as a monoplex reaction, to assess amplification efficiency; the human TNNT2 (Panel A) and GAPDH (Panel B) sequences were detected from five-fold serial dilutions of cdna (40ng to 2.6pg), using GoTaq Probe qpcr Master Mix for amplification (Panel A). The same pool of cdna was then used to evaluate performance of the GoTaq Probe qpcr Master Mix in amplifying the two assays as a duplex reaction (Panel C). 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 DNA polymerase, the thermal cycling 8 Schmidt, B. and Nassif, N.
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. Table 4. Average Cq s using the GoTaq Probe 2-Step RT-qPCR System with both Standard and FAST Master mixes. Average Cq s generated from triplicate reactions for each master mix are shown. cdna was generated from Human Reference Total RNA (Agilent: #750500), using the GoScript Reverse Transcription System. The GoTaq Probe qpcr Master Mix was used to amplify a human GAPDH TaqMan assay (Applied Biosystems: Hs02758991_g1; Vendor #2), using standard cycling on an AB 7500 instrument, and again using fast cycling on an AB 7500 FAST instrument. Standard curves were generated with both cycling methods to assess amplification efficiency; the human GAPDH sequence was detected from ten-fold serial dilutions of cdna (100ng to 0.1pg). The GoTaq Probe qpcr master mix demonstrated robust performance with both standard and fast cycling. The same pool of cdna was used to compare performance of the QuantiFast Probe Master Mix (Qiagen), which is branded specifically for fast cycling. 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. REFERENCES 1. Valasek, M.A. and Repa, J.J. (2005) The Power of Real-Time PCR Adv. Physiol. Educ. 29, 151 9. 2. 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. 3. Heid, C.A. et al. (1996) Real time quantitative PCR Genome Res. 6, 986 94. 4. Tyagi, S. and Kramer F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303. 5. Bessetti, J. (2007) An Introduction to PCR Inhibitors Profiles in DNA 10, 9 10. 6. Bustin, S.A. (2002) Quantification of mrna using real-time reverse transcription PCR (RT-PCR): Trends and problems. J. Mol. Endocrinol. 29, 23 39. 9 Schmidt, B. and Nassif, N.
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: http://www.promega.co.uk/resources/pubhub/optimized-reagents-forprobe-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. http://www.promega.co.uk/resources/pubhub/optimized-reagents-for-probe-based-qpcr-using-the-gotaqprobe-qpcr-and-rt-qpcr-systems/ Updated January 2014; tpub 115. Accessed Month Day, Year. For Research Use Only. Not for Use in Diagnostic Procedures. U.S. Pat. No. 6,242,235, Australian Pat. No. 761757, Canadian Pat. No. 2,335,153 and other patents and patents pending. Licensed under U.S. Pat. No. 5,587,287 and corresponding patents in other countries. For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: DISCLAIMER OF LICENSE No license is conveyed with the purchase of this product under any of US Pat. Nos. 5,210,015, 5,487,972, 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787 and 6,258,569 and corresponding patents outside the United States or any other patents or patent applications relating to the 5 Nuclease and dsdna-binding Dye Processes. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 USA. GoTaq is a registered trademark of Promega Corporation. TaqMan is a registered trademark of Roche Molecular Systems, Inc. 10 Schmidt, B. and Nassif, N.