IN THE BEGINNING

When the polymerase chain reaction (PCR) burst onto the scene in the mid-1980s, its usefulness for genetic analysis was immediately recognized. Indeed, the first publication of the PCR method was on its use in the prenatal diagnosis of sickle cell anemia. When the use of thermostable deoxyribonucleic acid (DNA) polymerases and programmable thermocyclers made PCR a commonly used method in the laboratory, the detection of genetic variation became a much easier enterprise. Instead of relying on laborious approaches such as restriction fragment length polymorphism (RFLP) analysis or DNA sequencing of complementary DNA clones to detect genetic variation, PCR allowed the “extraction” of a specific locus of the genome and produced sufficient quantities of it for further analysis. The main contributions of PCR to the detection of genetic variation are in three areas: amplification of small, unique regions of the genome harboring DNA sequence variants; discrimination of allelic differences between genomes; and amplification of products of other allelic discrimination reactions for detection by conventional means.

In the early days of the PCR revolution, the main obstacles to the deployment of PCR for genetic variation were the paucity of genomic sequence information for PCR primer design, the relatively high cost of oligonucleotide synthesis, and the laborious procedures used in DNA sequencing. Fortunately, automated DNA synthesis and DNA sequencing instruments became available in the early 1990s and the initial genomic mapping phase of the Human Genome Project provided the impetus to produce genetic markers based on PCR. As the speed of DNA sequencing and oligonucleotide synthesis increased while their cost went down, PCR became the principal approach to genetic analysis.

Alteration of microribonucleic acid (miRNA) expression in a disease compared to a healthy state and/or correlation of miRNA expression with clinical parameters (like disease progression or therapy response) may indicate that miRNAs can serve as clinically relevant biomarkers. An important first step for further functional characterization is the information about differential miRNA expression in cellular processes such as differentiation, proliferation, or apoptosis that may determine which disease-causing genes are specifically regulated by miRNAs or, vice versa, which genes regulate miRNA expression. Whatever question you would like to address, the precise information about the level of miRNA expression in a specific cell type or tissue is often considered an important first step. A range of methods can be used for the isolation and profiling of miRNAs. Two recent reviews on microRNA and quantitative polymerase chain reaction (PCR) in European Pharmaceutical Review addressed both topics individually in great detail but not their combination. This chapter aims to provide an insight into the application of quantitative real-time PCR (qPCR) to assay microRNA expression.

miRNA EXPRESSION PROFILING USING qPCR

miRNA-specific qPCR assays are frequently used to confirm data obtained from microarray experiments but can be used independently of course. On first glance, the major advantages of this technology over microarrays are (1) the speed of the assay, (2) the increased sensitivity with which miRNAs expressed even at low levels can be measured, (3) the extended dynamic range compared to microarray analysis, and (4) the low requirement for starting material (10 ng per reaction).

The PCR is widely used in many applications throughout the world. It has its secure place in the history of molecular biology as one of the most revolutionary methods ever. The principles of PCR are clear, but how can the reaction procedure be optimized to bring out the best in each assay? What is the status quo and what is next? Where are there areas for improvement?

INTRODUCTION

PCR is defined as a relatively simple heat-stable Taq polymerase–based technique, invented by Kary B. Mullis and coworkers, who were awarded the Nobel Prize for chemistry in 1993 for this discovery. However, this terrain is contested, and many other scientists were instrumental in making PCR work in all kinds of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein (immuno quantitative PCR [qPCR])–based applications. Reverse transcription (RT) followed by PCR represents a powerful tool for messenger RNA (mRNA) quantification. Nowadays, real-time RT–PCR is widely and increasingly used because of its high sensitivity, good reproducibility, and wide dynamic quantification range. Today, quantitative real-time RT–PCR (qRT–PCR) represents the most sensitive method for the detection and quantification of gene expression levels. It has its tremendous advantages in elucidating small changes in mRNA expression levels in samples with low RNA concentrations, from limited tissue samples and in single cell analysis. Sensitivity and reproducibility is a particular requirement of expression profiling, which focuses on the fully quantitative approach for mRNA quantification, rather than simply qualitative analysis.

ILLUMINATING THE UNSEEN

The polymerase chain reaction (PCR) is a revolutionary piece of chemistry that has upended the science of biology by allowing manipulation of individual molecules of deoxyribonucleic acid (DNA). With a broad reach into drug development, forensics, and the sequencing of the human genome, PCR has already touched persons who may never pick up a pipette. Relying upon awkward language like oligonucleotide and amplicon makes explanation of its mechanism a difficult lesson, but replicating any sequence of DNA is of fundamental importance to modern biotechnology.

And replicate it does. Not only does PCR succeed in finding the needle in the haystack, it proceeds to make an entire haystack out of needles! This unrelenting amplification of the DNA molecule evokes images of the Sorcerer's Apprentice, where the utility of a single broom is recognized and duplicated until it takes on a life of its own.

In certain laboratories, PCR can also wear out its welcome when the sorcery becomes hard to control. Replicating the very sequence that one intends to detect is both a blessing and a curse because residual molecules from previous experiments make it hard to start again with a clean slate. They can splash onto the laboratory bench, cling to gloves, and even launch into the air to leisurely float down onto an inconvenient location (inconvenient for the researcher, at least).

A BRIEF INTRODUCTION TO THE FEATURES OF RESPIRATORY VIRUSES AND THE ROLE OF POLYMERASE CHAIN REACTION IN DETECTING THEM

The principal detection of a virus in respiratory secretions tends to bestow upon it the colloquial title of a “respiratory virus.” Human respiratory viruses are the most numerous and most highly diverse, organ-defined group of viruses that we currently know of; not surprising, considering their efficient method of transmission. They infect with greater frequency (infections per person per year) and with a broader coverage (annual number of infected people worldwide) than does any other infectious agent. To date, most acute respiratory tract infectious entities are known to be viruses, and most of these have a monopartite ribonucleic acid (RNA) genome. Viruses, being what they are, intimately associate with human cells and secretions, from which they must be discriminated. This can make them difficult to detect, let alone characterize. Most of the modern focus on respiratory virus detection is now drawn by molecular methods. At the forefront of these, both in research and routine diagnostic laboratories, is the polymerase chain reaction (PCR), a technique lauded for its ability to detect a target from among a far superior number of nontarget sequences.

Data from the reinvigorated arena of respiratory virus research are pushing many new and old infectious disease issues to the forefront of microbiology, none more so than the question of what is required of our experimental design to determine whether a virus detected from the respiratory tract is the cause of respiratory symptoms.

Since the introduction of gene expression microarray technology, the number of applications and publications based on it has grown enormously. Nowadays, there is almost no institution or university in the field of molecular biology that has no genomic facility helping to apply this technique. Microarrays, an ordered assembly of thousands of probes, have the ability to allow the simultaneous determination of the expression levels of thousands of genes. This technique was used to describe gene programs that underlie various cellular processes, such as immunity and hormone responses, as well as to refine classifications of neoplasias, and to define diagnostic molecular markers for diseases. However, one drawback of the microarray technique is that, the more genes are tested, the higher the risk of identifying false positives as a result of random effects. Furthermore, biological and technical variations, including the microarray design, can affect the precision of microarray results. More difficult situations are found when working with complex multicellular tissue samples as compared to cell line experiments. The outcome of these microarray experiments can result in low fold changes and low signal intensities for differentially expressed genes, which makes it difficult to detect regulated genes reliably. Therefore, the identification of differentially expressed genes requires independent confirmation. Quantitative real-time reverse transcriptase–polymerase chain reaction (qPCR) is the method of choice because of its broad range of linearity, high sensitivity, and reproducibility and because it can be easily adapted to test several hundreds of transcripts.

The invention and successful practice of the polymerase chain reaction (PCR) by Kary Mullis and colleagues in 1983 set the stage for a scientific revolution. PCR established a base technology from which many specific and diverse applications have grown. PCR has played a crucial underlying technological role in many aspects of the genomic age that we experience today. The power to assess complete genomic sequences starting with minuscule amounts of target molecules entrenched PCR as the backbone of many subsequent analytical techniques. The sequencing of the genomes of many diverse species and the ability to discriminate individuals within a species have relied on PCR as an instrumental component.

The knowledge of genomes has led to the ability to identify sequences representing the coding genes that carry the blueprints for the construction of proteins. It is of great scientific interest to study the regulation of these gene-encoding messenger ribonucleic (mRNA) molecules. The study of gene expression has led to a better understanding of different biological states that exist within different tissue types, reflecting their different functions. Gene expression changes provide insight into underlying molecular and functional differences that exist between diseased and normal tissues. PCR has had a profound impact on gene expression studies as well. In 1991, while I was a junior scientist at Genentech, my scientific life was intensely affected by PCR. I was part of a team charged with developing assays to assess clinical outcomes of a vaccine treatment for human immunodeficiency virus (HIV) infections.

THERMOSTABLE ENZYMES

Many living organisms have been found in most challenging environments that most of us would not believe possible. These microorganisms, known as extremophiles, are found in most extreme conditions of salinity, alkalinity, pressure, temperature, and so on. Most of these microorganisms have been identified as members of domain archae, which are ancient living organisms. The utilization of these organisms and their components (including enzymes) has been studied for a number of applications.

To live in these extreme conditions, those microorganisms have cellular components including biocatalysts – enzyme proteins – that are active in such conditions. Many studies have been carried out to enrich knowledge about such enzymes by identifying and characterizing them. Thermostable enzymes present in microorganisms living in extremely high temperatures are the most extensively studied enzymes and have a number of industrial applications.

Organisms that grow at high temperatures are called thermophiles when they grow optimally between 50°C and 80°C and hyperthermophiles when they grow optimally between 80°C and 110°C. There are some organisms that can grow under extremely hot conditions up to 113°C. Such enzymes are typically not active if the temperature is less than 40°C. These enzymes are also useful in understanding enzyme evolution and molecular mechanisms for thermal stability of proteins and identification of upper temperature limits for enzyme functions.

The biological process of metastases requires multiple individual steps to successfully establish a solid tumor at a secondary site. Tumor cell(s) need to migrate through and from the primary tumor mass, intravasate into and survive within the hemopoietic or lymphatic vascular systems, extravasate from these systems into secondary tissues and initiate proliferation and angiogenesis. Multiple molecular and microenvironment factors influence these processes, defining which tumor cells survive, spread to distant organs and give rise to metastases. Despite considerable advances in the treatment of solid cancers, metastases remain the major clinical challenge for successful treatment. The classic view is that metastatic spread is a late process in disease progression. However, the prognosis for patients with small or even undetectable primary tumors is still limited by metastatic relapse, sometimes long after removal of the primary tumor. This has led to the hypothesis that primary tumors may shed tumor cells at an early stage, resulting in the dissemination of tumor cells to distant sites and development of metastases. Interestingly the bone marrow (BM) and lymph nodes (LN) seem to be common homing tissues for many different disseminating tumor cells, and so the early detection and characterization of tumor cells in these compartments could help guide treatment decisions before the onset of overt metastases, as well as in the setting of advanced disease. Unfortunately the number of tumor cells in these sites is usually small and they are not detected using current technologies.

CURRENT QUANTITATIVE POLYMERASE CHAIN REACTION USES IN QUANTITATIVE CLINICAL DIAGNOSTICS

Clinical diagnostics is progressively embracing the incorporation of translational medicine, as the diagnostic industry starts to lean toward personalized treatment. Direct benefits to clinical diagnostics will be achieved as a result of technical advancements providing unambiguous quantitative analysis of the transcriptome, although the quantitative clinical diagnostic sector is a relatively small part of the overall clinical diagnostic market. Interests in deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) detection and quantification have improved current knowledge of cell functions, including cell regulation, growth, expression markers, and transcription. The polymerase chain reaction (PCR) is one such research technique in mainstream clinical diagnostics that can provide quantitative analysis and assist in closing the “bench to bedside” gap found with translational medicine.

The reverse transcription–PCR (RT–PCR) is at present the most sensitive technique for mRNA detection, although quantitative RT–PCR (qRT–PCR) increasingly provides robust PCR product measurements from each cycle. Additionally, qPCR is commonly applied to clinical diagnostics, making the technique an industry standard for RNA product detection and quantification. An increase in qPCR applications, combined with the growing importance of clinical diagnostics, has permitted both areas to develop in parallel. Reflecting these advancements, the value of qPCR in quantitative clinical diagnostics has increased largely through the stochastic integration of fluorochrome that can be directly related to qualitative measurements. Qualitative end-point PCR measurements benefit from many of the detection strategies used in qPCR, but obviously lack quantitative application.

The qualitative and quantitative analysis of genetic material obtained from so-called “archival” tissue specimens (see next section for a definition of “archival”) is nowadays a routine operation in modern molecular laboratories thanks to improvements in the extraction methods of nucleic acids and subsequent amplification technology.

Today it is not only possible to analyze localized alterations like point mutations or the expression level of single genes in archival samples, but also to analyze large stretches of genomic material (up to several million base pairs of deoxyribonucleic acid [DNA] sequence) and to assess the expression level of thousands of genes in parallel. For nearly all applications, a polymerase chain reaction (PCR)-based amplification step of the extracted nucleic acid is necessary, and many investigations of this kind are virtually impossible without a powerful amplification technology like PCR. Therefore, “analysis of genetic material from archival specimens” is nearly synonymous with the title of this chapter.

First the word “archival” is explained a bit more in detail and the advantages of archival specimens in general are summarized. Then, several areas of basic and clinical research as well as routine diagnostics are described for which the ability to analyze genetic material extracted from archival specimens represents a major technological advancement, and that is aptly titled a “revolution.”

WHAT DOES “ARCHIVAL” MEAN?

The term “archival” in the title of this chapter has a specific meaning in the context of processing and storing tissue samples from human beings or animals for the purpose of histopathological examination, but it is also used in both a somewhat broader sense and a much broader sense.

Advances in science and technology come at an ever increasing pace. What was state of the art can be obsolete in just a few years. The old saw “The work that earns a Nobel prize today will be a graduate thesis ten years from now” may underestimate the rate of change. The hard-earned accomplishments of scientists and technologists can be made to seem trivial well within the time frame of a career. Faced with this, what do scientists celebrate as their accomplishments?

In the Stephen Sondheim play Sunday in the Park with George, about the pioneering artist, George Seurat, the character Dot says that the only things worth “passing on” to posterity are “children and art.” I agree wholeheartedly with the children part but I wondered about how broadly art could be defined in this context. As evidenced by university colleges still organized around “Arts and Sciences” – a holdover I believe from classical uses of the terms – science and art did not used to be so far apart. I believe that beautiful, inventive thinking can be art, and good science is full of beautiful, inventive thinking – such as PCR.

When I first heard of PCR, I thought – art. When I later joined Cetus and heard from Kary Mullis his idea to use a thermostable enzyme – art. When Kary proposed the Hot Start (a bit arcane but to someone who was now an aficionado) – art.