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A Multiplex Real-Time PCR-Platform Integrated into Automated Extraction Method for the Rapid Detection and Measurement of Oncogenic HPV Type-Specific Viral DNA Load from Cervical Samples
The persistent infection with most frequent high-risk (HR)-HPV types (HPV-16, -18, -31, -33, -45, -52, and -58) is considered to be the true precursor of neoplastic progression. HR-HPV detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer, although also the HR-HPV DNA load is considered an ancillary marker for persistent HPV infection. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Absolute Quantification of Viral DNA: The Quest for Perfection
In spite of the impressive technical refinement of the PCR technology, new-generation real-time PCR assays still suffer from two major limitations: the impossibility to control both for PCR artifacts (with the important caveat of false-negative results) and for the efficiency of nucleic acid recovery during the preliminary extraction phase of DNA from the biological sample. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter
Mediator probe PCR (MP PCR) is a novel detection format for real-time nucleic acid analysis. Label-free mediator probes (MP) and fluorogenic universal reporter (UR) oligonucleotides are combined to accomplish signal generation. Compared to conventional hydrolysis probe PCRs costs can thus be saved by using the same fluorogenic UR for signal generation in different assays. This tutorial provides a practical guideline to MP and UR design. MP design rules are very similar to those of hydrolysis probes. The major difference is in the replacement of the fluorophore and quencher by one UR-specific sequence tag, the mediator. Fur...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

mRNA and microRNA Purity and Integrity: The Key to Success in Expression Profiling
RNA quality control is a crucial step in guaranteeing integer nondegraded RNA and receiving meaningful results in gene expression profiling experiments, using micro-array, RT-qPCR (Reverse-Transcription quantitative PCR), or Next-Generation-Sequencing by RNA-Seq or small-RNA Seq. Therefore, assessment of RNA integrity and purity is very essential prior to gene expression analysis of sample RNA to ensure the accuracy of any downstream applications. RNA samples should be nondegraded or fragmented and free of protein, genomic DNA, nucleases, and enzymatic inhibitors. Herein we describe the current state-of-the-art RNA quality...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Introduction to Digital PCR
Digital PCR (dPCR) is a molecular biology technique going through a renaissance. With the arrival of new instrumentation dPCR can now be performed as a routine molecular biology assay. This exciting new technique provides quantitative and detection capabilities that by far surpass other methods currently used. This chapter is an overview of some of the applications currently being performed using dPCR as well as the fundamental concepts and techniques this technology is based on. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Selection of Reliable Reference Genes for RT-qPCR Analysis
Reference genes have become the method of choice for normalization of qPCR data. It has been demonstrated in many studies that reference gene validation is essential to ensure accurate and reliable results. This chapter describes how a pilot study can be set up to identify the best set of reference genes to be used for normalization of qPCR data. The data from such a pilot study should be analyzed with dedicated algorithms such as geNorm to rank genes according to their stability—a measure for how well they are suited for normalization. geNorm also provides insights into the optimal number of reference genes and the ...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Minimum Information Necessary for Quantitative Real-Time PCR Experiments
The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines were published in 2009 with the twin aims of providing a blueprint for good real-time quantitative polymerase chain reaction (qPCR) assay design and encouraging the comprehensive reporting of qPCR protocols. It had become increasingly clear that variable pre-assay conditions, poor assay design, and incorrect data analysis were leading to the routine publication of data that were often inconsistent, inaccurate, and wrong. The problem was exacerbated by a lack of transparency of reporting, with the details of technical information ina...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Twenty Years of qPCR: A Mature Technology?
Quantitative PCR is the “gold standard” technology to quantify nucleic acids and, since the first report describing real-time PCR detection in 1993, its use has been grown exponentially. More recent technological advancements have extended the field of applications ranging from high-resolution melting detection to digital PCR. Nowadays, it is a very accessible technique, but some pitfalls should be overcome in order to achieve robust and reliable analysis. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Developing Noninvasive Diagnosis for Single-Gene Disorders: The Role of Digital PCR
Cell-free fetal DNA constitutes approximately 10 % of the cell-free DNA found in maternal plasma and can be used as a reliable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD) from early pregnancy. The relatively high levels of maternal background can make detection of paternally inherited point mutations challenging. Diagnosis of inheritance of autosomal recessive disorders using qPCR is even more challenging due to the high background of mutant maternal allele. Digital PCR is a very sensitive modified method of quantitative real-time PCR (qPCR), allowing absolute quantitation and rare allele det...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Clinical Applications Using Digital PCR
Molecular diagnostics and disease-specific tailored treatments are now being introduced to patients at many hospitals and clinics throughout the world (Strain and Richman, Curr Opin HIV AIDS 8:106–110, 2013) and becoming prevalent in the nonscientific literature. Instead of generically using a “one treatment fits all” approach that may have varying levels of effectiveness to different patients, patient-specific molecular profiling based on the genetic makeup of the disease and/or a more accurate pathogen titer could provide more effective treatments with fewer unwanted side effects. (Source: Springer prot...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Posttranscriptional Regulatory Networks: From Expression Profiling to Integrative Analysis of mRNA and MicroRNA Data
Protein coding RNAs are posttranscriptionally regulated by microRNAs, a class of small noncoding RNAs. Insights in messenger RNA (mRNA) and microRNA (miRNA) regulatory interactions facilitate the understanding of fine-tuning of gene expression and might allow better estimation of protein synthesis. However, in silico predictions of mRNA–microRNA interactions do not take into account the specific transcriptomic status of the biological system and are biased by false positives. One possible solution to predict rather reliable mRNA-miRNA relations in the specific biological context is to integrate real mRNA and miRNA tr...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Gene Expression Analysis by qPCR in Clinical Kidney Transplantation
Patients with a kidney transplant may encounter chronic dysfunction of their graft. Once damage in the graft has established, therapeutic intervention is less efficient. Clinical parameters and morphologic evaluation of biopsies are used for determining diagnosis and prognosis of the patient. Quantitative polymerase chain reaction (qPCR) may be integrated in clinical practice to facilitate routine diagnostics, risk assessment with respect to graft outcome, and determination of the response to therapy by the patient. The success of qPCR assays is highly dependent on the adequacy of the methodological procedures performed. H...
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Circulating Cell-Free DNA in Cancer
We describe a method to accurately quantify total cfDNA in plasma and our particular approach to the measurement of tumor deriving cfDNA. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

A Reliable Assay for Rapidly Defining Transplacental Metastasis Using Quantitative PCR
To choose the most appropriate treatment for children affected by a transplacental metastasis, it is crucial to ascertain the maternal origin of the tumor. Up-to-date conclusive diagnosis is generally achieved through fluorescence in situ hybridization or karyotyping analysis. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news

Real-time Quantification Assay to Monitor BCR-ABL1 Transcripts in Chronic Myeloid Leukemia
The BCR-ABL1 fusion gene, the causative lesion of chronic myeloid leukemia (CML) in >95 % of newly presenting patients, offers both a therapeutic and diagnostic target. Reverse-transcription quantitative polymerase chain reaction technology (RT-qPCR), utilizing primer–probe combinations directed to exons flanking the breakpoint junctional region, offers very high levels of both specificity and sensitivity, in a scalable, robust, and cost-effective assay. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news