Since its introduction in the 1980s, the polymerase chain reaction (PCR) has revolutionized how we diagnose diseases and investigate their underlying causes at the genetic level. Moreover, variants on the original technique have unlocked the door to both qualitative and quantitative gene expression analysis. Reverse transcriptase polymerase chain reaction (RT-PCR) is a highly sensitive, specific technique for studying differential gene expression, and real-time or quantitative reverse transcriptase PCR (real-time RT-PCR) provides valuable quantitative data that allows the user to see how much a gene has been activated or repressed. By profiling genes involved in specific pathways, diseases, or processes, one can put together a clear picture of the changes occurring in the body, potentially leading to discovery of diagnostic and prognostic markers to enhance both curative and preventative medicine.
The mechanics of PCR
PCR exponentially amplifies small quantities of DNA. The technique utilizes thermal cycling to accomplish three key steps: denaturation, annealing, and extension. A DNA template is mixed with a thermostable DNA polymerase such as Taq, free deoxynucleotide triphosphate molecules (dNTPs), and short oligonucleotides called primers, which are complementary for the beginning and end of the sequence to be amplified. In the denaturation step, double-stranded (ds)DNA separates into single strands, and in the annealing step, primers bind to their complementary sequences. DNA polymerase then produces complementary DNA strands in the extension step. The size and the purity of the final DNA product can be analyzed using gel electrophoresis.
PCR and genetic testing
In addition to being well established in basic science and translational research laboratories, PCR is also commonly used for several clinical applications. Genetic testing can reveal mutations responsible for hereditary diseases or for oncogenic variants, allowing a medical professional to make informed decisions about treatment, and helping prospective parents know whether their children will be at risk for developing certain diseases. Additionally, PCR can be used to detect small numbers of infectious microbes such as viruses and bacteria, permitting earlier or more accurate diagnosis, as well as screening of blood donations.
Two variants on the classic PCR technique have expanded its potential applications, making it indispensable for gene expression analysis in research settings. These variants are reverse transcription PCR (RT-PCR) and real-time PCR. RT-PCR makes use of reverse transcriptase enzymes to convert messenger RNA (mRNA) into complementary DNA (cDNA). PCR is then performed on the cDNA, permitting qualitative analysis of which genes are expressed and repressed in certain cells or tissues.
Real-time PCR uses fluorescent markers to quantify, with each cycle, how much dsDNA is being produced. Real-time RT-PCR therefore permits quantitative analysis of gene expression, showing the extent to which genes are up- or down-regulated. There are two common types of marker used in real-time PCR. Dyes such as SYBR® Green bind to all dsDNA and fluoresce upon binding, whereas fluorescent probes bind to specific DNA sequences and fluoresce upon cleavage by Taq polymerase during the extension step of PCR.
In recent years, real-time RT-PCR arrays have emerged to provide quantitative data on the expression of many related genes at one time, allowing researchers a comprehensive overview of the changes occurring in a system. For example, RT2 Profiler PCR Arrays are real-time RT-PCR assays for 84 individual genes that have been organized into pathway-focused arrays based on extensive bioinformatic analysis and text mining. This type of analysis is especially useful in research areas such as cancer, drug toxicity, and inflammation, where the expression of many genes can be altered at once.
PCR and drug toxicity
Toxicity is a major concern in the testing of new potential drugs, and real-time RT-PCR arrays provide a rapid, comprehensive overview of how cells respond to these compounds. Arikawa and colleagues demonstrated the effectiveness of RT2 Profiler PCR Arrays in distinguishing between a diabetes drug that was discontinued due to high liver toxicity, troglitazone (Tro), previously sold as Rezulin, and two other drugs still on the market, rosiglitazone (Rosi, sold as Avandia®) and pioglitazone (Pio, sold as ACTOS®).1
Arikawa et al treated HepG2 liver cells with each of the drugs, and then studied gene expression using the Human Stress & Toxicity PathwayFinder PCR Array and the Human Drug Metabolism PCR Array. Both arrays yielded different gene expression profiles for Tro than for Rosi and Pio, demonstrating the ability of real-time RT-PCR to predict, corroborate, and suggest explanations for clinical observations. In particular, the Human Stress & Toxicity PathwayFinder PCR Array showed significantly higher expression levels of the genes HSPA6, CRYAB, and CSF2 with Tro treatment (Figure 1).
Moreover, gene expression relating to drug metabolism was also altered. The Drug Metabolism PCR Array revealed a 7.1-fold increase in induction of GSTP1 with Tro, as well as a 297.5 fold-change compared to controls in expression of MT2A. By contrast, GSTP1 expression was unchanged with Rosi or Pio, and these drugs up-regulated MT2A by only 8.1- and 18.6-fold, respectively (Table 1). By analyzing gene expression changes, the team was able to demonstrate significant differences in a cell system model, between a drug that humans can tolerate and a drug with unsafe toxicity levels.
PCR and cancer biomarkers
Cancer cells show significantly altered gene expression compared to normal cells, and real-time RT-PCR is therefore a highly suitable technique for discovering tumor biomarkers and potential therapeutic targets. Arikawa et al used the Human Cancer PathwayFinder PCR Array to compare breast cancer tissue with normal breast tissue in order to identify which genes showed altered expression after transformation.1 This array includes representative genes from the following biological pathways involved in tumorigenesis: adhesion, angiogenesis, apoptosis, cell cycle control, cell senescence, DNA damage repair, invasion, metastasis, signal transduction molecules, and transcription factors. Comparison of normal and tumor tissue with this array showed that 24 genes were differentially regulated by at least threefold between normal and tumor samples, suggesting potential targets to investigate for future therapies.
To confirm and extend these findings, the team used a second, unmatched breast tissue sample with the Human Extracellular Matrix and Adhesion Molecules PCR Array, as six of the genes showing altered expression in the first sample related to this pathway. They found 38 genes with differential expression compared to normal tissue, and four of these changed expression in the same direction as the unmatched breast cancer sample that had been used for the Human Cancer PathwayFinder PCR Array. Performing arrays on unmatched samples and finding similar results suggests that alteration in these genes may be a general marker of breast tissue transformation. Finding consistent changes among cancers from a statistically significant number of different individuals is the essence of biomarker discovery.
In summary, RT-PCR and real-time RT-PCR are powerful tools to study changes in gene expression. They have already proven their utility for clinical applications as well as yielded fantastic discoveries in the research lab. Pathway-focused analysis will continue to clarify many biological processes, and PCR technology will continue to advance. In addition to the advent of PCR arrays for mRNA, PCR arrays for somatic mutations, DNA methylation, and miRNA have also begun to emerge, such as qBiomarker Somatic Mutation PCR Arrays, EpiTect® Methyl qPCR Arrays, and miScript® miRNA PCR Arrays. Ultimately, PCR technology will be one of the keys to achieving the goals of personalized medicine and “bench-to-bedside” application of research discoveries through its ease of use, cost-effectiveness, and molecular power. In the future, medical advances will likely make use of PCR-based approaches for gene expression to yield early clues for diagnosis and prognosis, and to monitor the effectiveness of treatments.
Trademarks EpiTect®, miScript® (QIAGEN Group); Avandia® (a registered trademark used under license by GlaxoSmithKline Inc.); Actos® (Takeda Pharmaceutical Co.); SYBR® (Life Technologies Corporation).
Disclaimer
RT2 Profiler PCR Arrays, qBiomarker Somatic Mutation PCR Arrays, EpiTect® Methyl qPCR Arrays, and miScript® miRNA PCR Arrays are intended for molecular biology applications.
These products are not intended for the diagnosis, prevention, or treatment of a disease.
Allison Bierly is a technical writer at QIAGEN in Frederick, MD. She received her PhD from Cornell University in 2009, studying the immune response to parasitic infection, and went on to a post-doctoral fellowship in molecular immunology at the National Cancer Institute. She joined QIAGEN in 2011, where she writes about assay technologies with a focus on pathway biology.
References
- Arikawa E, et al. RT2 Profiler PCR Array application examples: pathway-focused gene expression profiling in toxicology, oncology, and immunology research. http://www.sabiosciences.com/manuals/PCRArrayWhitePaper_App.pdf.
Accessed March 20, 2012.
