A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Advances in the biologic sciences and technology are providing molecular targets for diagnosis and treatment of cancer. Lymphoma is a group of cancers with diverse clinical courses. Gene profiling opens new possibilities to classify the disease into subtypes and guide a differentiated treatment. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. For accurate gene expression profiling by real-time PCR, several parameters must be considered and carefully validated. These include the use of reference genes and compensation for PCR inhibition in data normalization. Quantification by real-time PCR may be performed as either absolute measurements using an external standard, or as relative measurements, comparing the expression of a reporter gene with that of a presumed constantly expressed reference gene. Sometimes it is possible to compare expression of reporter genes only, which improves the accuracy of prediction. The amount of biologic material required for real-time PCR analysis is much lower than that required for analysis by traditional methods due to the very high sensitivity of PCR. Fine-needle aspirates and even single cells contain enough material for accurate real-time PCR analysis. <s> BIB001 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Discovered just over a decade ago, microRNA (miRNA) is now recognized as one of the major regulatory gene families in eukaryotic cells. Hundreds of miRNAs have been found in animals, plants and viruses, and there are certainly more to come. Through specific base-pairing with mRNAs, these tiny approximately 22-nt RNAs induce mRNA degradation or translational repression, or both. Because a miRNA can target numerous mRNAs, often in combination with other miRNAs, miRNAs operate highly complex regulatory networks. In this article, we summarize the current status of miRNA gene mining and miRNA expression profiling. We also review up-to-date knowledge of miRNA gene structure and the biogenesis mechanism. Our focus is on animal miRNAs. <s> BIB002 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> BACKGROUND ::: Real-time (RT) PCR methods for discovering and genotyping single nucleotide polymorphisms (SNPs) are becoming increasingly important in various fields of biological sciences. SNP genotyping is widely used to perform genetic association studies aimed at characterising the genetic factors underlying inherited traits. The detection and quantification of somatic mutations is an important tool for investigating the genetic causes of tumorigenesis. In infectious disease diagnostics there is an increasing emphasis placed on genotyping variation within the genomes of pathogenic organisms in order to distinguish between strains. ::: ::: ::: METHODS ::: There are several platforms and methods available to the researcher wishing to undertake SNP analysis using real-time PCR methods. These use fluorescent technologies for discriminating between the alternate alleles of a polymorphism. There are several real-time PCR platforms currently on the market. Two of the key technical challenges are allele discrimination and allele quantification. ::: ::: ::: CONCLUSIONS ::: Applications of this technology include SNP genotyping, the sensitive detection of somatic mutations and infectious disease subtyping. <s> BIB003 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Background ::: As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. <s> BIB004 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput. <s> BIB005 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases. <s> BIB006 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs. <s> BIB007 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis. <s> BIB008 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Copy number changes are known to be involved in numerous human genetic disorders. In this context, qPCR-based copy number screening may serve as the method of choice for targeted screening of the relevant disease genes and their surrounding regulatory landscapes. qPCR has many advantages over alternative methods, such as its low consumable and instrumentation costs, fast turnaround and assay development time, high sensitivity and open format (independent of a single supplier). In this chapter we provide all relevant information for a successfully implement of qPCR-based copy number analysis. We emphasize the significance of thorough in silico and empirical validation of the primers, the need for a well thought-out experiment design, and the importance of quality controls along the entire workflow. Furthermore, we suggest an appropriate and practical way to calculate copy numbers and to objectively interpret the results. The provided guidelines will most certainly improve the quality and reliability of your qPCR-based copy number screening. <s> BIB009 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> DNA copy number variants (CNVs) have been reported in many human diseases including autism and schizophrenia. Primary Open Angle Glaucoma (POAG) is a complex adult-onset disorder characterized by progressive optic neuropathy and vision loss. Previous studies have identified rare CNVs in POAG; however, their low frequencies prevented formal association testing. We present here the association between POAG risk and a heterozygous deletion in the galactosylceramidase gene (GALC). This CNV was initially identified in a dataset containing 71 Caucasian POAG cases and 478 ethnically matched controls obtained from dbGAP (study accession phs000126.v1.p1.) (p = 0.017, fisher's exact test). It was validated with array comparative genomic hybridization (arrayCGH) and realtime PCR, and replicated in an independent POAG dataset containing 959 cases and 1852 controls (p = 0.021, OR (odds ratio) = 3.5, 95% CI -1.1-12.0). Evidence for association was strengthened when the discovery and replication datasets were combined (p = 0.002; OR = 5.0, 95% CI 1.6-16.4). Several deletions with different endpoints were identified by array CGH of POAG patients. Homozygous deletions that eliminate GALC enzymatic activity cause Krabbe disease, a recessive Mendelian disorder of childhood displaying bilateral optic neuropathy and vision loss. Our findings suggest that heterozygous deletions that reduce GALC activity are a novel mechanism increasing risk of POAG. This is the first report of a statistically-significant association of a CNV with POAG risk, contributing to a growing body of evidence that CNVs play an important role in complex, inherited disorders. Our findings suggest an attractive biomarker and potential therapeutic target for patients with this form of POAG. <s> BIB010 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Genetic analysis is one of the fastest-growing areas of clinical diagnostics. Fortunately, as our knowledge of clinically relevant genetic variants rapidly expands, so does our ability to detect these variants in patient samples. Increasing demand for genetic information may necessitate the use of high throughput diagnostic methods as part of clinically validated testing. Here we provide a general overview of our current and near-future abilities to perform large-scale genetic testing in the clinical laboratory. First we review in detail molecular methods used for high throughput mutation detection, including techniques able to monitor thousands of genetic variants for a single patient or to genotype a single genetic variant for thousands of patients simultaneously. These methods are analyzed in the context of pharmacogenomic testing in the clinical laboratories, with a focus on tests that are currently validated as well as those that hold strong promise for widespread clinical application in the near future. We further discuss the unique economic and clinical challenges posed by pharmacogenomic markers. Our ability to detect genetic variants frequently outstrips our ability to accurately interpret them in a clinical context, carrying implications both for test development and introduction into patient management algorithms. These complexities must be taken into account prior to the introduction of any pharmacogenomic biomarker into routine clinical testing. <s> BIB011 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> In recent years, quantitative real-time PCR tests have been extensively developed in clinical microbiology laboratories for routine diagnosis of infectious diseases, particularly bacterial diseases. This molecular tool is well-suited for the rapid detection of bacteria directly in clinical specimens, allowing early, sensitive and specific laboratory confirmation of related diseases. It is particularly suitable for the diagnosis of infections caused by fastidious growth species, and the number of these pathogens has increased recently. This method also allows a rapid assessment of the presence of antibiotic resistance genes or gene mutations. Although this genetic approach is not always predictive of phenotypic resistances, in specific situations it may help to optimize the therapeutic management of patients. Finally, an approach combining the detection of pathogens, their mechanisms of antibiotic resistance, their virulence factors and bacterial load in clinical samples could lead to profound changes in the care of these infected patients. <s> BIB012 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches. Considering the diverse technologies now available for transcriptome analysis, methods for standardising measurements between platforms will be paramount if their diagnostic impact is to be maximised. Therefore, the use of RNA standards and other reference materials is also discussed. <s> BIB013 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> Molecular biomarkers found their way into many research fields, especially in molecular medicine, medical diagnostics, disease prognosis, risk assessment but also in other areas like food safety. Different definitions for the term biomarker exist, but on the whole biomarkers are measureable biological molecules that are characteristic for a specific physiological status including drug intervention, normal or pathological processes. There are various examples for molecular biomarkers that are already successfully used in clinical diagnostics, especially as prognostic or diagnostic tool for diseases. Molecular biomarkers can be identified on different molecular levels, namely the genome, the epigenome, the transcriptome, the proteome, the metabolome and the lipidome. With special "omic" technologies, nowadays often high throughput technologies, these molecular biomarkers can be identified and quantitatively measured. This article describes the different molecular levels on which biomarker research is possible including some biomarker candidates that have already been identified. Hereby the transcriptomic approach will be described in detail including available high throughput methods, molecular levels, quantitative verification, and biostatistical requirements for transcriptional biomarker identification and validation. <s> BIB014 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> MicroRNAs (miRNAs) in human saliva have recently become an emerging field in saliva research for -diagnostics applications and its potential role in biological implications. miRNAs are short noncoding RNA molecules that play important roles in regulating a variety of cellular processes. Dysregulation of miRNAs are known to be associated with many diseases. miRNAs were found present in the saliva of OSCC patients and could serve as potential biomarkers for oral cancer detection. Understanding the biological function of miRNAs in association with diseases is important towards utilizing miRNAs as diagnostic markers. There are currently a variety of profiling methods available for detecting miRNA expression levels. In this chapter, we overview the Applied Biosystem Stem-loop RT based Taqman MicroRNA Assay for salivary miRNA profiling. Using this highly sensitive and specific assay, miRNAs in saliva are profiled with only a few nanograms of starting RNA. This method is also applicable for studying biomarkers in other body fluids or clinical samples that contain small amounts of RNA. <s> BIB015 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> MicroRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes. Cell-free miRNAs measured in blood plasma have emerged as specific and sensitive markers of physiological processes and disease. In this study, we investigated whether circulating miRNAs can serve as biomarkers for the detection of autologous blood transfusion, a major doping technique that is still undetectable. Plasma miRNA levels were analyzed using high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after autologous blood transfusion (blood bag storage time 42 days) in 10 healthy subjects and 10 controls without transfusion. Other serum markers of erythropoiesis were determined in the same samples. Our results revealed a distinct change in the pattern of circulating miRNAs. Ten miRNAs were upregulated in transfusion samples compared with control samples. Among these, miR-30b, miR-30c, and miR-26b increased significantly and showed a 3.9-, 4.0-, and 3.0-fold change, respectively. The origin of these miRNAs was related to pulmonary and liver tissues. Erythropoietin (EPO) concentration decreased after blood reinfusion. A combination of miRNAs and EPO measurement in a mathematical model enhanced the efficiency of autologous transfusion detection through miRNA analysis. Therefore, our results lay the foundation for the development of miRNAs as novel blood-based biomarkers to detect autologous transfusion. <s> BIB016 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Applications of qPCR <s> The diagnosis of cardiomyopathies by endomyocardial biopsy analysis is the gold standard for confirmation of causative reasons but is failing if a sample does not contain the area of interest due to focal pathology. Biopsies are revealing an extract of the current situation of the heart muscle only, and the need for global organ-specific or systemic markers is obvious in order to minimize sampling errors. Global markers like specific gene expression signatures in myocardial tissue may therefore reflect the focal situation or condition of the whole myocardium. Besides gene expression profiles, microRNAs (miRNAs) represent a new group of stable biomarkers that are detectable both in tissue and body fluids. Such miRNAs may serve as cardiological biomarkers to characterize inflammatory processes, to confirm viral infections, and to differentiate various forms of infection. The predictive power of single miRNAs for diagnosis of complex diseases may be further increased if several distinctly deregulated candidates are combined to form a specific miRNA signature. Diagnostic systems that generate disease-related miRNA profiles are based on microarrays, bead-based oligo sorbent assays, or on assays based on real-time polymerase chain reactions and placed on microfluidic cards or nanowell plates. Multiparametric diagnostic systems that can measure differentially expressed miRNAs may become the diagnostic tool of the future due to their predictive value with respect to clinical course, therapeutic decisions, and therapy monitoring. We discuss here specific merits, limitations and the potential of currently available analytical platforms for diagnostics of heart muscle diseases based on miRNA profiling. Contains 34 references. <s> BIB017
qPCR can be applied for several applications spanning a wide range of use cases. The most commonly used application is gene expression analysis. In addition to quantification experiments, qPCR can be used for the analysis of copy number variations, DNA methylation, and genomic variants. 3.1. Quantification of DNA, RNA, and DNA methylation qPCR was originally designed for DNA quantification and has been extensively developed in clinical microbiology laboratories for routine diagnosis of infectious diseases. It is applied for detecting bacterial, fungal, and viral pathogens, especially when testing to discover the source of an unknown infection BIB012 . In addition to DNA quantification, it has been widely used to quantify the expression of messenger RNA (mRNA) BIB005 . First, RNA is reverse transcribed into a cDNA template, which is then used in qPCR reactions to detect and quantify gene expression products. Amongst others, it has been successfully applied for the analysis of transcriptional biomarkers BIB013 BIB014 , cancer BIB001 , and Mendelian diseases BIB010 . Since their discovery in 1993, microRNAs (miRNAs) have been a valuable research target for scientist worldwide. miRNAs are small, regulatory, noncoding single-stranded RNAs, which are usually 20-25 nucleotides long BIB002 . The first qPCR assay to quantify miRNAs was developed almost a decade ago BIB006 , and subsequent protocols for using relative and absolute quantification have been developed BIB007 . miRNAs have been successfully identified as possible biomarkers for cancer BIB015 , autologous transfusion BIB016 , diabetes BIB006 and cardiomyopathies BIB017 . In addition to miRNAs, a large number of different non-coding RNAs have been described, where many show regulatory functions. Amongst others, long noncoding RNAs (lncRNAs), small inhibiting RNA (siRNA), and small nuclear RNA (snRNA) can be quantified by qPCR methods. DNA methylation is a heritable epigenetic modification in genomes which is known to be involved in biological processes such as regulation of gene expression, cell differentiation, DNA structure and disease states BIB008 . To date, several protocols for determining the methylation state have been developed such as real-time Quantitative Methylation-Specific PCR (QMSP) BIB004 , methylationsensitive restriction enzyme digestion PCR (MSRE-PCR) BIB009 , MethyLight BIB003 , and methylation-sensitive high resolution melting (MS-HRM) BIB011 . The analysis of methylation qPCR data depends on the selected protocol and involves absolute or relative quantification as well as high resolution melting (HRM) analysis.