Improved productivity and throughput in qPCR without compromising performance

The technique is widely used in SNP genotyping, mutation detection and quantification, and gene dosage studies. When coupled with a reverse transcription step, quantitative RT-PCR (qRT-PCR) is the method of choice for detection and quantification of gene expression and expression profiling. Broad adaptation of qPCR has been enabled by the development of less expensive, high quality real-time PCR thermal cyclers and analysis software that improve the accuracy of detection and simplify data analysis; as well as stabilized hot-start master mix reagents that simplify reaction assembly and improve qPCR assay performance and accuracy.

The recent introduction of fast-ramp rate qPCR instruments tailored for 384-well format offer high assay throughput and shorter assay times. Beyond 384-well high density plates, other efforts are under way for 1536-well and high density array based formats. There is a constant desire on the part researchers, particularly those involved in pharmaceutical drug discovery efforts, to maximize real-time PCR throughput at significantly faster speeds than existing instrumentation. The increased throughput and ability to scan whole genome or global gene expression profiles will have broad implications across drug development industry and accelerate introduction of new therapeutic compounds.

Increasing throughput by using higher density formats with reduced individual reaction volumes has advantages and potential pitfalls. The benefits include simultaneous assay of hundreds of loci or genes and reduced cost of reagents. However, reduced assay volumes also decreases the overall number of DNA or RNA template copies in the assay, potentially reducing sensitivity and /or precision of detection and quantification. 

The potential benefit of instruments that support fast cycling conditions is predicated on the assumption that available reagents support fast cycling without compromising the amplification efficiency, dynamic range or sensitivity. In addition, the reduced sample volume required for fast cycling requires reagent systems that can effectively amplify from few template copies of RNA or DNA. While PCR amplification technology has been shown to be very effective, amplification from reduced number of template copies is by no means routine for every sample or every assay. These assay parameters require development of reagent systems that are highly specific and have rapid kinetics to allow fast cycling with high degree of sensitivity and reliability.

PerfeCta™ qPCR FastMixes™ have been specifically developed to address these needs for either SYBR® Green or TaqMan® probe-based detection chemistries. These reagents enable high efficiency qPCR results with significantly faster protocols for increased productivity (Table 1). PerfeCta qPCR FastMixes are 2X concentrated, ready-to-use reaction cocktails that deliver maximum PCR efficiency, sensitivity, specificity and robust fluorescent signal. FastMixes can be used with fast cycling protocols on either rapid ramp rate or conventional block real-time cyclers (Table 2).  PerfeCta qPCR FastMixes are available for all real-time PCR instrument platforms.

Table 1. qPCR Cycling Comparison

An important component of all the PerfeCta™ FastMixes is AccuFast™ Taq DNA polymerase. This hot-start thermostable polymerase was developed to allow instantaneous activation at 95ºC. It contains a proprietary mixture of monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step (up to 48 hours at room temperature). When heated to 95ºC, these antibodies are irreversibly inactivated during the initial PCR denaturation step. This rapid-release automatic hot-start helps maximize specificity in qPCR. Highly specific amplification is crucial to successful qPCR, particularly when using SYBR Green I dye technology as this dye binds to and detects any dsDNA generated during amplification. However, even probe-based qPCRs can be adversely affected by non-specific amplification since these products compete for substrates and can impair qPCR efficiency.

Rapid recovery of fully active, unmodified Taq DNA polymerase is critical for efficient extension kinetics. Replication of fragments up to 200 bp is complete in less than 20s at 60ºC. It is important to note that while chain extension may actually be completed in much less than 20s, the minimum data collection time varies for different real-time PCR systems. This dwell time must be taken into consideration when choosing a particular cycling protocol to allow accurate collection and analysis of the fluorescent signal.

The physical properties of the intended DNA target are another factor that must be considered in designing a fast cycle program. Optimal initial denaturation time is template dependent and will affect qPCR efficiency and sensitivity. 20 - 30s at 95ºC is effective for most applications. However, amplification of genomic DNA or supercoiled plasmid DNA targets may require 5 to 10 min at 95ºC to fully denature and fragment the template. This minimizes the potential for renaturation of long fragments and/or repetitive sequence regions that can impair PCR. Short double-stranded DNA template (PCR product) or single-stranded DNA template, such as cDNA template generated using the qScript™ cDNA Synthesis Kit or qScript cDNA SuperMix, may require as little as 1s at 95ºC. The stability of RNA: DNA duplex present in first-strand product should be considered when programming the denaturation step, particularly if using oligo-dT primed cDNA with an RNase H deficient reverse transcriptase.

SYBR® Green FastMixes:
PerfeCta SYBR Green FastMixes, are provided as 2X concentrated, ready-to-use reaction cocktails that contains all components, except primers and template for real-time quantitative PCR on a variety of real-time PCR systems (See Table 2). This unique combination of proprietary buffer, stabilizers, and AccuFast™ Taq DNA polymerase delivers maximum PCR efficiency, sensitivity, specificity and robust fluorescent signal using fast, or conventional, cycling protocols with SYBR Green qPCR.

qPCR FastMixes for probe-based detection:
PerfeCta qPCR FastMixes are 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers, probe(s), and template for real-time quantitative. An added benefit offered by qPCR FastMixes is the inclusion of dUTP and uracil-N-glycosylase (UNG) to prevent amplification of carry-over contamination from previous dU-containing PCRs. During the development of this product we found superior fast-cycling performance with dUTP-containing formulations, particularly for MGB-modified TaqMan probes, as compared to equivalent dTTP mixes. The proprietary buffer, stabilizers and AccuFast Taq DNA polymerase have been specifically optimized to deliver maximum PCR efficiency, sensitivity, and robust fluorescent signal with TaqMan® or TaqMan MGB probe chemistry when using rapid PCR cycle times and reduced reaction volumes. This affords greater reagent economy and laboratory throughput on conventional or rapid ramp rate qPCR systems. The enhanced specificity of this FastMix suppresses cross-reactivity between homologous sequences, improving detection and discrimination in SNP applications.

Instrument compatibility
Different real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. PerfeCta qPCR FastMixes and PerfeCta SYBR Green FastMixes have been formatted to work optimally in all real-time PCR systems (Table 2).

Table 2. PerfeCta FastMix Instrument Compatibility Guide

FAST qRT-PCR:
Another aspect of real-time PCR is expression profiling and quantification of RNA or mRNA. One of the methods involved in quantification of RNA is one-step RT-PCR in which reverse transcription of RNA is immediately followed by PCR amplification of cDNA in the same reaction. While this method was introduced more than 12 years ago, optimization of conditions for maximal reverse transcription and maximum PCR efficiency has been difficult. This is especially true for quantitative applications of this technology using TaqMan probes. The nature of TaqMan probe chemistry has profound effects on the efficiency of RT-PCR amplification. Different TaqMan probe chemistries demand specific conditions for optimal qRT-PCR. Quanta BioSciences has developed two separate kits specifically optimized for use with TaqMan MGB probes or conventional 5’ hydrolysis probes using TAMRA™, Black Hole Quencher™ or BHQplus™ quencher.

qScript™ One-Step Fast MGB qRT-PCR kits
TaqMan probes modified with minor grove binder (MGB) moieties offer a number of advantages over conventional probe chemistries. These probes form more stable duplexes allowing use of significantly shorter probes. They are also quenched more efficiently and are more specific in various applications such as single base mismatch detection. However, the use of MGB modified probes can present unique challenges for effective one-step qRT-PCR, generally resulting in lower sensitivity. qScript One-Step Fast MGB qRT-PCR Kits have been specifically optimized to provide exceptional qRT-PCR efficiency and sensitivity with TaqMan-MGB probes, such as TaqMan Gene Expression Assays from Applied Biosystems. The novel composition of the One-Step Fast MGB Master Mix  maximizes the activities of both the reverse transcriptase and Taq DNA polymerase to allow unbiased amplification of low copy transcripts in the presence of high copy reference genes. These kits are suitable for use in duplexed one-step qRT-PCR with VIC™-MGB labeled TaqMan Endogenous Controls, other TaqMan MGB probes.

qScript™ One-Step Fast qRT-PCR kit
The qScript™ one Step Fast qRT-PCR kits have been developed for rapid and highly sensitive qRT-PCR of a wide range of RNA targets. These kits are ideal for use with the conventional 5’ hydrolysis probes using TAMRA, Black Hole, or Black Hole plus quencher. The novel composition of the qScript One-Step Fast Master Mix maximizes the activities of both the reverse transcriptase and Taq DNA polymerase to allow unbiased amplification of low copy transcripts in the presence of high copy reference genes. These kits produce highly sensitive results using rapid cycling protocols but can also be used with conventional real-time thermocyclers using slower protocols. The qScript™ One-Step Fast qRT-PCR Kits include a 4X concentrated master mix to allow analysis of larger volumes of sample. This results in ability to quantify rare target sequences in dilute samples.

In conclusion, availability of new generation of fast qPCR instruments tailored for high-density sample analysis offer high assay throughput and shorter assay times. Use of these instruments with specific reagents for qPCR or qRT-PCR that allow efficient and robust amplification of target RNA or DNA can result in improved laboratory productivity (Fig 1). Perfecta qPCR FastMixes and qScript Fast One-Step qRT-PCR kits have been specifically designed to function optimally with these instruments. The increased throughput and ability to scan whole genome or global gene expression profiles will have broad implications across all areas of research.

Figure 1. Improved productivity on standard or Fast-Block qPCR Systems
 
Quanta BioSciences is an innovative developer of reagents for genomic and genetic research. Founding members of Quanta BioSciences led the development of pioneering reagents including Invitrogen’s SuperScript® RT-PCR Kits, Platinum® Taq and Bio-Rad's iScript™ and iQ™/iTaq™ SuperMixes. For unique client needs, Quanta’s Custom development team works directly with clients to develop specific solutions. For more information please visit www.quantabio.com or e-mail sales@quantabio.com.