Challenges and advancements in QPCR analysis

The technology is conceptually simple, easy to use, and widely applicable. However, there are still a host of challenges that affect the accuracy of this highly sensitive method. These challenges range from sample preparation, the use of reference standards to data analysis, and validation.

Stratagene supports QPCR researchers in every aspect; a broad portfolio of QPCR reagents and an affordable QPCR analysis system with user-friendly software for data analysis provide a complete solution approach to real-time QPCR.

Sample preparation for QPCR analysis

RNA stabilisation from cells
Most researchers are faced with sample preparation challenges. The isolation of RNA from multiple samples is tedious, time consuming, and often results in variable yields. Stratagene has developed a cell lysis buffer technology, SideStep lysis and stabilisation buffer, to eliminate RNA loss and immediately stabilise nucleic acids for use in quantitative reverse transcription-PCR (QRT-PCR).

The SideStep method employs a patent pending buffer system, allowing a researcher to completely avoid nucleic acid purification and proceed directly to real-time QPCR detection, mRNA extraction, and cDNA synthesis.

Additionally, small cell input applications benefit from the SideStep lysis method because RNA loss is eliminated. This method is ideal for siRNA knockdown validation, miRNA detection, and mRNA profiling among a host of other cell-based applications. By skipping the nucleic acid isolation step, the SideStep lysis and stabilisation buffer allows access to the entire nucleic acid complement by QPCR without biasing gene expression. Direct QRT-PCR from SideStep lysates has shown equivalent yields when compared to column-purified formats.

RNA purification from paraffin-embedded samples
Archived tissue specimens are rich sources of material for extensive analysis of mRNA expression utilising QRT-PCR. However, the chemical nature of fixatives and fixation time significantly affects the quality of RNA extracted from these tissues.

Using a standard proteinase K digestion protocol is ineffective at reversing the RNA modifications caused by the fixation process, resulting in poor and unreliable RNA recovery. Stratagene has developed the Absolutely RNA FFPE kit to completely reverse the RNA modifications and, thus, more quality RNA is isolated from difficult paraffin-embedded tissue sections for QPCR analysis.

Purify RNA from laser microdissected cells
Reliable recovery of RNA from small cell populations, such as laser micro-dissection (LMD) cells, is also particularly challenging to analyse by QRT-PCR. Laser micro-dissection is a technique used to isolate a pure subpopulation of cells from tissue sections containing a heterogeneous mixture for detailed molecular analysis. Isolating high-quality total RNA from LMD-recovered cells can be challenging and inefficient when standard methods are used.

To address these small sample challenges, the Absolutely RNA microprep and nanoprep kits effectively isolate pure RNA from small samples, while avoiding ethanol precipitation. Moreover, the Absolutely RNA nanoprep kit is the only kit optimised to isolate RNA from a single cell.

cDNA Synthesis for QPCR

Another challenge for QPCR researchers is to synthesize cDNA for QPCR analysis. Since most commercial first strand cDNA synthesis kits are developed for cloning applications, they lack the linearity and sensitivity required for QPCR detection. The StrataScript QPCR cDNA synthesis kit is designed for high-efficiency conversion of RNA to cDNA and is fully optimised for real-time QPCR applications.

The high sensitivity obtained with this kit is due to the inclusion of the QPCR-grade StrataScript Reverse Transcriptase and a high-efficiency cDNA synthesis buffer. In addition, the StrataScript QPCR cDNA synthesis kit is formatted as a master mix that reduces pipetting steps and enhances reproducibility in two-step QRT-PCR. Furthermore, it employs a fast, 15-minute protocol for most targets.

Highly sensitive detection of DNA or RNA using SYBR® Green

The Brilliant SYBR Green QPCR and QRT-PCR reagents provide a universal solution to real-time QPCR detection and gene quantification, exhibiting greater sensitivity compared to other SYBR Green kits. The SYBR Green dye binds to any PCR product and therefore does not require the use of sequence-specific probes. All Brilliant reagent kits contain the SureStart Taq DNA Polymerase, a hot-start version of Taq that minimises amplification of non-specific PCR products.

Sensitive and specific probe-based detection of up to four multiplex targets

The Brilliant probe-based QPCR and QRT-PCR reagents are compatible with sequence-specific probes including TaqMan probes, molecular beacons, and Scorpions. These reagents offer a wide linear dynamic range of amplification for up to two simultaneous targets. These QRT-PCR kits contain StrataScript reverse transcriptase and are available in one-step and two-step formats. A passive reference dye is included for versatility and to maximise performance on different instrument platforms.

The Brilliant Multiplex QPCR master mix allows researchers to amplify up to four targets in a single reaction. Each multiplex reaction requires far less template than if the targets were analysed in separate reactions to lower the overall costs of routine analysis. Also, it maximises the use of precious or rare samples, such as those obtained from biopsies or laser micro-dissection. Importantly, the sensitivity remains equivalent to that seen in singleplex. Thus, researchers can maximise the use of each precious sample without sacrificing sensitivity. The Brilliant multiplex QPCR master mix also provides sufficient reaction components to accurately quantify both low- and high-abundance targets in the same tube. This enables more successful multiplexing without concern for bias due to abundance level.

QPCR and QRT-PCR directly from cells without purification

Conducting rapid and precise real-time analysis directly from cell lysates is now possible with the Brilliant SideStep QPCR and QRT-PCR kits. The Brilliant SideStep QPCR and QRT-PCR reagents take advantage of the new SideStep buffer technology to ensure accurate gene expression data by minimising the loss or degradation of nucleic acid molecules by going directly from cells to a real-time QPCR amplification. Ideally, RT-PCR is performed using primers designed to span intron-exon boundaries. Genomic DNA, miRNA, and RNA can all be measured directly from SideStep lysates. RNA is stable in the lysate for up to six months, which is ideal for a host of cell-based gene expression applications such as mRNA profiling and siRNA knockdown validation.

Fast and economical detection of DNA or RNA using SYBR® Green

The FullVelocity SYBR Green QPCR and QRT-PCR master mixes use the novel, high speed FullVelocity DNA polymerase to provide rapid and efficient real-time quantification of DNA or cDNA. Using a fast cycling protocol with a combined annealing/extension step, the polymerase allows 40-cycle amplification to be completed in less than one hour on most real-time instrument platforms. In addition, the FullVelocity DNA polymerase is less prone to inhibition by SYBR Green I than Taq DNA polymerase, promoting greater efficiency. Since these master mixes provide rapid and efficient quantification of RNA in a convenient format, they are the ideal choice when reproducibility and throughput are of primary concern.

QPCR reference standards

In order to reliably compare data across multiple experiments and instruments, it is essential to have a constant reference material to assess the performance of each QPCR reaction and quantify gene expression levels. The QPCR Reference Total RNAs are high-quality, universal RNA controls for quantitative PCR gene-expression analysis. These RNAs are extracted from quality cell lines representing different human or mouse tissues, pooled together for maximal gene representation. The broad gene coverage makes the RNA reference material ideal to be used as a universal reference for nearly any human or mouse gene being investigated in QPCR experiments. These high-quality human or mouse total RNA controls are manufactured for maximum representation of low, medium, and high-abundant gene transcripts. Furthermore, the QPCR reference total RNAs are optimised for QPCR and tested to be free of genomic DNA.

Cutting edge performance with flexible and affordable QPCR systems

Researchers need a QPCR system, but are unsure which features are most useful to support their current research goals and future research efforts. In addition, the transition to real-time QPCR on a limited budget can be challenging. Ideally, researchers need a high-performance QPCR system loaded with features and flexibility to support a wide range of applications at an affordable price. The first QPCR system to meet this need is the Mx3000P QPCR system. The Mx3000P system was designed to include all the features of the most advanced QPCR instruments, but at an affordable price for the individual researcher getting started in QPCR.

As QPCR technology advances and applications for the technology rapidly expand, researchers also need a QPCR system with flexibility to support any application or QPCR chemistry. The new Mx3005P QPCR system offers unmatched flexibility and capability to support the rapid advancement of QPCR technology. The advanced optical system design of the Mx3005P system supports detection of five dyes simultaneously and custom optical configurations to support any QPCR chemistry.

Real-time QPCR technology allows a researcher to generate more comprehensive data sets in less time compared to traditional nucleic acid quantification techniques. Therefore, the most important feature of any QPCR system is the user interface and data analysis software package. The MxPro QPCR Software featured on Mx systems combines the most important aspects of QPCR software with an intuitive user interface, advanced data analysis algorithms and flexible data reporting. The majority of time and effort in QPCR is spent performing data analysis and generating reports. The MxPro software is designed to streamline these operations and generate the highest quality data.

Summary

Stratagene provides a complete solution approach to real-time quantitative PCR (QPCR), simplifying the challenges researchers face in sample preparation and data analysis. For novice researchers, they will be benefit from the premixed reagent kits and turnkey solution provided by the Mx QPCR System and the MxPro QPCR Software. Meanwhile, more experienced QPCR users can appreciate the flexibility of reagent kits that support user customisation and demanding assay optimisation, as well as the powerful and yet user-friendly software.