Biomolecular Detection and Quantification

Over the last few decades, there has been a decline in Northern bobwhite quail ( Colinus virginianus ) throughout their native range. While there are various factors that may be influencing this decline, it is suggested that parasites should be taken into consideration as a potential contributor in the Rolling Plains Ecoregion. High prevalence of the eyeworm ( Oxyspirura petrowi ) and caecal worm ( Aulonocephalus pennula ) in bobwhite of this region, coupled with a continuous decline, creates a need to assess infection through alternative methods for regional surveillance...

mRNA is a critical biomolecule involved in the manifestation of the genetic code into functional protein molecules. Its critical role in the central dogma has made it a key target in many studies to determine biomarkers and drug targets for numerous diseases. Currently, there is a growing body of evidence to suggest that RNA molecules around the size of full-length mRNA transcripts can be assayed in the supernatant of human urine and urinary extracellular mRNA could provide information about transcription in cells of urogenital tissues...

The kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®. Using a rapid deletion mutant of Taq (KlenTaq™ ), about 1 s was required for every 70 bp of product length...

[This corrects the article DOI: 10.1016/j.bdq.2018.12.003.].

Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing...

The standard approach for quantitative estimation of genetic materials with qPCR is calibration with known concentrations for the target substance, in which estimates of the quantification cycle ( Cq ) are fitted to a straight-line function of log( N 0 ), where N 0 is the initial number of target molecules. The location of Cq for the unknown on this line then yields its N 0 . The most widely used definition for Cq is an absolute threshold that falls in the early growth cycles. This usage is flawed as commonly implemented: threshold set very close to the baseline level, which is estimated separately, from designated "baseline cycles...

[This corrects the article DOI: 10.1016/j.bdq.2018.12.001.].

Non-coding RNAs were established in the last decade as a new valuable biomarker class for human diseases. Specifically, circular RNAs (circRNAs) were only recently discovered as a new large group of non-coding RNAs that, due to their circular configuration, are metabolically more stable compared to their linear counterparts and therefore highly suitable for biomarker use. Based on high-throughput sequencing, the catalogs of endogenous circRNAs with disease relevance and correlation continue to grow steadily...

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed...

No abstract text is available yet for this article.

An increasing number of studies demonstrate the potential use of cell-free DNA (cfDNA) as a surrogate marker for multiple indications in cancer, including diagnosis, prognosis, and monitoring. However, harnessing the full potential of cfDNA requires (i) the optimization and standardization of preanalytical steps, (ii) refinement of current analysis strategies, and, perhaps most importantly, (iii) significant improvements in our understanding of its origin, physical properties, and dynamics in circulation. The latter knowledge is crucial for interpreting the associations between changes in the baseline characteristics of cfDNA and the clinical manifestations of cancer...

Direct detection of single stained DNA fragments in flow is a very sensitive method for nucleic acid detection which does not need any amplification process. We have developed an instrument for direct counting and sizing of single DNA fragments (single or double stranded DNA) in flow with integrated sample volume measurement for concentration determination. As the method is a potential reference method for DNA quantification, processes affecting the measurement uncertainty are of major interest. Additionally, comparison of this method to the orthogonal method of digital PCR is useful with the restriction of low specificity of the direct detection method...

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching...

Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition...

Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons...

[This corrects the article DOI: 10.1016/j.bdq.2015.10.001.].

The RNA-to-cDNA conversion step in transcriptomics experiments is widely recognised as inefficient and variable, casting doubt on the ability to do quantitative transcriptomics analyses. Multiple studies have focused on ways to optimise this process, resulting in contradictory recommendations. Here we explore the problem of reverse transcription efficiency using digital PCR and the RT method's impact on subsequent data analysis. Using synthetic RNA standards, an example experiment is presented, outlining a method to (1) determine relevant efficiency and variability values and then to (2) incorporate this information into downstream analyses as a way to improve the accuracy of quantitative transcriptomics experiments...

The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase...

Objectives: Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples...

Amplification curves from quantitative Real-Time PCR experiments typically exhibit a sigmoidal shape. They can roughly be divided into a ground or baseline phase, an exponential amplification phase, a linear phase and finally a plateau phase, where in the latter, the PCR product concentration no longer increases. Nevertheless, in some cases the plateau phase displays a negative trend, e.g. in hydrolysis probe assays. This cycle-to-cycle fluorescence decrease is commonly referred to in the literature as the hook effect ...