April 1, 2009 (Vol. 29, No. 7)

A Plethora of qPCR Products Facilitate Cutting-Edge Research

The development of real-time quantitative polymerase chain reaction (qPCR) as a technique for measuring the abundance of specific sequences of DNA or RNA in a sample has revolutionized biotech and clinical research arenas.

Over the last few years, improvements and developments in qPCR technology, reagents, and automation have broadened routine use of the technology. Applications for qPCR span areas including gene-expression analysis, genotyping, SNP analysis, viral load testing, pathogen detection, drug target validation, and RNA interference analysis.

The use of qPCR to analyze miRNAs is, in principle, another major application for the technology, but the small size of miRNAs means traditional qPCR approaches are problematic, reported Peter Mouritzen, Ph.D., director of product development at Exiqon.

“miRNAs play key roles in the post-transcriptional control of gene expression and regulation of many biological processes. They have been implicated in a wide range of pathologies, from cancer to neurodegenerative diseases, and may represent promising new targets for therapeutic intervention or as biomarkers for disease diagnosis and prognosis. Mature miRNAs, however, are usually only 18–22 nucleotides long. Positioning two qPCR primers, themselves typically 18–20 nucleotides, along this length is not really feasible using traditional approaches, and shorter primers have melting temperatures (Tm) below a useful optimum.”

To address this problem, Exiqon has developed the miRCURY LNATM microRNA PCR system, a SYBR green qPCR platform for mature miRNA quantitation. The technology, reviewed at the qPCR symposium in Munich, hinges on the use of primers into which locked nucleic acids (LNAs) are incorporated.

LNA nucleosides contain a methylene bridge that spans the ribose sugar, essentially locking the nucleotide into an ideal conformation for Watson-Crick binding. LNA-containing oligos such as primers demonstrate increased binding affinity and specificity, Dr. Mouritzen continued. “Using LNAs we can make shorter qPCR primers with minimal or no sequence overlap, minimizing the risk of primer-dimer formation. LNAs also significantly increase primer binding affinity, raise the Tm by 2–8°C per LNA monomer incorporated, and increase primer specificity to the point where single nucleotide mismatches can be distinguished.”

The two-step miRCURY qPCR process involves first converting miRNA into cDNA by reverse transcription using a miRNA-specific primer. This is then amplified by qPCR using the miRNA-specific short LNA primer. Exiqon’s research has shown that assays utilizing LNA-based primers allow detection of low expression miRNAs from a single cell (10 pg of total RNA), demonstrate a high dynamic range (accurate quantitation over a range of 8 logs), and a lower detection limit of just 10 copies per cell, according to Dr. Mourizen.

Multiplex Approach

As our understanding of the human genome starts to yield more information about the interplay of genes in disease progression and development, the ability to multiplex quantitative nucleic acid analysis technologies has become a goal for technological development, reported Jim Thorn, Ph.D., European bioseparations product manager at Beckman Coulter. “While qPCR has remained the backbone of microarray validation studies and SNP detection research, its drawback is that it can only handle one gene per test, and multiplexing the qPCR process has so far remained elusive, ” said Dr. Thorn.

For studies where only a few genes or transcripts need to be analyzed, the one-gene-one-assay approach may not be such a problem, but when large numbers of genes need to be evaluated in copious samples, the costs can escalate. “High-throughput qPCR instrumentation is now standard in many large laboratories, but if the requirement is to look at a signature of, for example, 30 genes in 1,000 samples, you’d need to run 30,000 tests, and then triple that to carry out three technical replications. The costs in terms of reagent requirements can become untennable.”

To address these issues, Beckman Coulter has commercialized a capillary electrophoresis-based system, the GenomeLab GeXP genetic analysis system, as a quantitative platform that utilizes a patented, multiplexed reverse transcription PCR (RT-PCR) approach to investigate multiple genes or gene sets.

The GenomeLab GeXP is essentially an endpoint PCR reaction coupled with a separation technology, which can run up to 40 genes per sample for gene expression, Dr. Thorn explained. “It’s a capillary electrophoresis sequencer, enabled to separate gene-expression samples. So when you run the assay you see a separate signal for each gene. For applications in gene-expression analysis, we supply the user with the GeXP software to design multiplex assays and the relevant GeXP kits for gene-specific reverse transcription and PCR amplification of their target mRNA from 25 ng of total RNA.”

The multiplexed endpoint PCR reaction is carried out on a standard PCR platform. This part of the technology hinges on a universal priming strategy, such that the PCR reaction uses four primer types. Two per gene are the forward and reverse gene-specific primers carrying a universal tail, which the customer obtains from a preferred provider. The other two primers are universal primers that bind to those tails and are the same for all genes in the multiplex.

“The chemistry is set up so there is a huge excess of the universal primers. This means that for the first one or two PCR cycles the gene-specific primers kick in, and after that the universals take over. Effectively this results in a PCR reaction that locks the ratios of the genes at an early stage, so when the universal primers take over replication, the target sequences are always copied in the same ratio. We have a reaction that behaves as if it’s one single type of PCR product, even though there could be up to 40 or so different genes being amplified.”

After amplification, a small sample of the product is transferred into a 96-well plate for separation and analysis in the GenomeLab GeXP. “Importantly, while microarray and qPCR-based technologies can typically investigate twofold or greater changes in gene expression, the GenomeLab GeXP technology can reliably detect expression changes down to as low as 0.5-fold,” Dr. Thorn claimed.

Quality Control

New technologies aside, skilled use of qPCR is essential if meaningful and robust results are to be obtained, stressed Steffen Müller, Ph.D., senior field application scientist at Agilent Technologies. “About 60–70% of the customers I deal with use qPCR in gene-expression analysis, and most of the other 30–40% work in pathogen detection. Yet while it is common practice to test the quality of a sample that goes into a microarray experiment, quality control (QC) of any sort in qPCR is often considered less important.”

This omission of QC in qPCR, both in terms of actual sample quality and assay quality (including robustness and reproducibility), is largely because of the comparatively low cost of the technology, Dr. Müller added. “Most labs would just repeat their qPCR experiments if they fail, especially if their sample was not in short supply. However, users need to understand that in many cases their experiments won’t fail completely due to poor QC, but will give results that significantly under- or overestimate the quantities of DNA or RNA that are really present. And as researchers strive to detect smaller and smaller quantities of nucleic acids, this need for quality control becomes even more crucial.”

Potential points of variance in the qPCR workflow are manifold, Dr. Müller noted. These range from the quality of the actual sample (impacted by degradation of nucleic acids, presence of inhibitors, or co-purified salts) and efficiency of sample extraction to reverse transciption efficiency, type of enzyme/priming method, and even plasticware and reagents. “All these factors can influence the results of a qPCR experiment.”

To address quality control at different stages of the qPCR process, Agilent offers the microfluidics-based 2100 Bioanalyzer for the analysis of DNA, RNA, proteins, and cells. “In gene-expression research, for example, running the prepared RNA sample through the Bioanalyzer will provide an electropherogram of individual RNAs, and also generate an RNA integrity number (RIN). The Agilent RIN-algorithm provides a user-independent score of quality and unambiguous assessment of RNA integrity for any measured sample.”
Knowing the quality of the RNA will then allow better amplicon design. “In my experience, many messenger sequences are degraded from the 5´ end, so if a sample of RNA is highly degraded, it might be an idea to move the amplicon for qPCR toward the 3´ end of the messenger. Without checking sample quality first, this aspect of design would be impossible.”

A sample quality test should be followed by a pilot study to assess the specificity of the assay itself, Dr. Müller continued. “Melting curve profiles using SYBR green assays are not specific because there are so many variables influencing the shape of the melt curve, and they also give no idea of product size. Most sizing information is, therefore, derived from PCR products on slab gels. The resolution of a slab gel, however, may not be high enough to detect the presence of two amplicons if the size of the unspecific amplicon is similar to that of the desired molecule. Running a sample of the PCR product through the Bioanalyzer will detect the presence of any unspecific amplicons, and help verify the PCR result.”

According to TriLink BioTechnologies, its CleanAmp Precision PCR Primers enhance results in multiplex, one-step RT-PCR when compared to unmodified PCR primers.

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