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We review different methodologies to estimate the expression levels of microRNAs (miRNAs) using real-time quantitative PCR (qPCR). As miRNA analysis is a fast changing research field, we have introduced novel technological approaches and compared them to existing qPCR profiling methodologies. qPCR also remains the method of choice for validating results obtained from whole-genome screening (e.g. with...
Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements...
Quantitative qPCR is a routinely used method for the accurate quantification of nucleic acids. Yet it may generate erroneous results if the amplification process is obscured by inhibition or generation of aberrant side-products such as primer dimers. Several methods have been established to control for pre-processing performance that rely on the introduction of a co-amplified reference sequence, however...
Background: High resolution melting (HRM) is an emerging new method for interrogating and characterizing DNA samples. An important aspect of this technology is data analysis. Traditional HRM curves can be difficult to interpret and the method has been criticized for lack of statistical interrogation and arbitrary interpretation of results. Methods: Here we report the basic principles and first applications...
The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted...
Circulating Tumour Cells (CTCs) can be released from the primary tumour into the bloodstream and may colonize distant organs giving rise to metastasis. The presence of CTCs in the blood has been documented more than a century ago, and in the meanwhile various methods have been described for their detection. Most of them require an initial enrichment step, since CTCs are a very rare event. The different...
High resolution melting is a new method of genotyping and variant scanning that can be seamlessly appended to PCR amplification. Limitations of genotyping by amplicon melting can be addressed by unlabeled probe or snapback primer analysis, all performed without labeled probes. High resolution melting can also be used to scan for rare sequence variants in large genes with multiple exons and is the...
Experiments using quantitative real-time PCR to test hypotheses are limited by technical and biological variability; we seek to minimise sources of confounding variability through optimum use of biological and technical replicates. The quality of an experiment design is commonly assessed by calculating its prospective power. Such calculations rely on knowledge of the expected variances of the measurements...
Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. Unfortunately, what many users fail to appreciate is that numerous critical issues in the workflow need to be addressed before biologically meaningful and trustworthy conclusions can be drawn. Here, we review the entire workflow from the planning...
Circulating nucleic acids are present in the blood of humans and other vertebrates. During the last 10 years researchers actively studied cell-free nucleic acids present in plasma or serum with great expectations of their use as potential biomarkers for cancer and other pathologic conditions. In the present manuscript the main findings related to the principal characteristics of circulating nucleic...
The next-generation DNA sequencing workflows require an accurate quantification of the DNA molecules to be sequenced which assures optimal performance of the instrument. Here, we demonstrate the use of qPCR for quantification of DNA libraries used in next-generation sequencing. In addition, we find that qPCR quantification may allow improvements to current NGS workflows, including reducing the amount...
MicroRNAs (miRNAs) are small (∼22nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating...
Given the highly dynamic nature of mRNA transcription and the potential variables introduced in sample handling and in the downstream processing steps (Garson et al. (2009) [4]), a standardized approach to each step of the RT-qPCR workflow is critical for reliable and reproducible results. The MIQE provides this approach with a checklist that contains 85 parameters to assure quality results that will...
Advances in qPCR technology allow studies of increasingly large systems comprising many genes and samples. The increasing data sizes allow expression profiling both in the gene and the samples dimension while also putting higher demands on sound statistical analysis and expertise to handle and interpret its results. We distinguish between exploratory and confirmatory statistical studies. In this paper...
The correlation of gene and protein expression changes in biological systems has been hampered by the need for separate sample handling and analysis platforms for nucleic acids and proteins. In contrast to the simple, rapid, and flexible workflow of quantitative PCR (qPCR) methods, which enable characterization of several classes of nucleic acid biomarkers (i.e. DNA, mRNA, and microRNAs), protein...
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...
For real-time monitoring of PCR amplification of DNA, quantitative PCR (qPCR) assays use various fluorescent reporters. DNA binding molecules and hybridization reporters (primers and probes) only fluoresce when bound to DNA and result in the non-cumulative increase in observed fluorescence. Hydrolysis reporters (TaqMan® probes and QZyme™ primers) become fluorescent during DNA elongation and the released...
The polymerase chain reaction has facilitated the ready analysis of nucleic acids. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The production of aqueous droplets in a two phase flow is shown to be readily...
Quantitative real-time PCR (qPCR) is a frequently used, sensitive and accurate method to study gene expression profiles. However, its throughput was so far limited for routine laboratories to 384 reactions per run based on the limitations of the available instruments. Recently, the LightCycler® 1536 Instrument was launched providing a high-throughput solution for qPCR with the analysis of 1536 reactions...
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