Biomolecular Detection and Quantification最新文献

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences (HMG-I/Y, FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.

Since the discovery of microRNAs (miRNAs), circulating miRNAs have been proposed as biomarkers for disease. Consequently, many groups have tried to identify circulating miRNA biomarkers for various types of diseases including cardiovascular disease and cancer. However, the replicability of these experiments has been disappointingly low. In order to identify circulating miRNA candidate biomarkers, in general, first an unbiased high-throughput screen is performed in which a large number of miRNAs is detected and quantified in the circulation. Because these are costly experiments, many of such studies have been performed using a low number of study subjects (small sample size). Due to lack of power in small sample size experiments, true effects are often missed and many of the detected effects are wrong. Therefore, it is important to have a good estimate of the appropriate sample size for a miRNA high-throughput screen. In this review, we discuss the effects of small sample sizes in high-throughput screens for circulating miRNAs. Using data from a miRNA high-throughput experiment on isolated monocytes, we illustrate that the implementation of power calculations in a high-throughput miRNA discovery experiment will avoid unnecessarily large and expensive experiments, while still having enough power to be able to detect clinically important differences.

Reverse Transcription quantitative real-time PCR (RT-qPCR) is applied to quantify gene transcript levels in a wide range of investigations. Proper assessment of RNA integrity is essential for reliable assessment of gene expression levels, as RNA molecules are acutely vulnerable to degradation. However, RNA quality control measures are still infrequently reported in rat toxicological studies, which impede proper evaluation of gene expression data reliability. The high operational cost of microfluidic capillary electrophoresis systems along with paucity of alternative methods for the quantitative assessment of rat RNA integrity constitute potential hurdles to the systematic implementation and reporting of RNA integrity assessment in rat studies. This manuscript describes the adaptation of an alternative RT-qPCR-based 3′:5′ assay as an additional option for the quantitative assessment of rat RNA integrity. Two PCR primer sets were designed on the 3′ and 5′ regions of a rat housekeeping gene to evaluate RNA integrity by measuring the relative expression (3′:5′ ratio) of these amplicons. The 3′:5′ ratios were then compared to Agilent Bioanalyzer’s RNA integrity number (RIN) for a wide range of RNA samples originating from different tissues, cultured cell lines and rat strains that were prepared freshly, stored for years at −80 °C, purchased commercially or intentionally degraded. The 3′:5′ ratios and RIN values presented similar assessment of RNA integrity status from intact to heavily degraded samples. Based on the LOWESS regression of this large comparison dataset, 3′:5′ ratio threshold criteria equivalent to RIN cut-off values can be proposed for the selection of RNA samples for RT-qPCR analyses. This qPCR-based assay is easy to implement, cost-effective, and provides a reliable quantification of RNA integrity to assist in the selection of rat RNA samples suitable for downstream RT-qPCR gene expression analyses.

Human health and safety depend on reliable measurements in medical diagnosis and on tests that support the selection and evaluation of therapeutic intervention and newly discovered molecular biomarkers must pass a rigorous evaluation process if they are to be of benefit to patients. Measurement standardization helps to maximize data quality and confidence and ultimately improves the reproducibility of published research. Failure to consider how a given experiment may be standardized can be costly, both financially as well as in time and failure to perform and report pre-clinical research in an appropriately rigorous manner will hinder the development of diagnostic methods. Hence standardization is a crucial step in maintaining the integrity of scientific studies and is a key feature of robust investigation.

Monitoring of minimal residual disease (MRD) has become an important clinical aspect for early relapse detection during follow-up care after cancer treatment. Still, the sensitive detection of single base pair point mutations via Next-Generation Sequencing (NGS) is hampered mainly due to high substitution error rates. We evaluated the use of NGS for the detection of low-level variants on an Ion Torrent PGM system. As a model case we used the c.1849G > T (p.Val617Phe) mutation of the JAK2-gene. Several reaction parameters (e.g. choice of DNA-polymerase) were evaluated and a comprehensive analysis of substitution errors was performed. Using optimized conditions, we reliably detected JAK2 c.1849G > T VAFs in the range of 0.01–0.0015% which, in combination with results obtained from clinical data, validated the feasibility of NGS-based MRD detection. Particularly, PCR-induced transitions (mainly G > A and C > T) were the major source of error, which could be significantly reduced by the application of proofreading enzymes. The integration of NGS results for several common point mutations in various oncogenes (i.e. IDH1 and 2, c-KIT, DNMT3A, NRAS, KRAS, BRAF) revealed that the prevalent transition vs. transversion bias (3.57:1) has an impact on site-specific detection limits of low-level mutations. These results may help to select suitable markers for MRD detection and to identify individual cut-offs for detection and quantification.

With the rapid advances in next generation sequencing (NGS) technologies, clinical and public health microbiology laboratories are increasingly adopting NGS technology in their workflows into their existing diagnostic cycles. In this bacteriology focused review, we review aspects and considerations for applying NGS in the clinical microbiology settings, and highlight the impact of such implementation on the analytical and post-analytical stages of diagnosis

Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful. Consequently, poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range. We present an overview of the main steps in the primer design workflow, with data that illustrate some of the unexpected variability that often occurs when theory is translated into practice. We also strongly urge researchers to report as much information about their assays as possible in their publications.

Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to C_q or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template −plasmid DNA- and non-template −mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow.

Introduction

For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms.

Materials and methods

RNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method.

Results

Both ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments.

Conclusions

Our study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.

Background

O’Neill’s recent Review on Antimicrobial Resistance expressed the view that by 2020 high-income countries should make it mandatory to support antimicrobial prescribing with rapid diagnostic evidence whenever possible.

Methods

Routine microbiology diagnosis of 95 respiratory specimens from patients with severe infection were compared with those generated by the Unyvero P55 test, which detects 20 pathogens and 19 antimicrobial resistance markers. Supplementary molecular testing for antimicrobial resistance genes, comprehensive culture methodology and 16S rRNA sequencing were performed.

Results

Unyvero P55 produced 85 valid results, 67% of which were concordant with those from the routine laboratory. Unyvero P55 identified more potential pathogens per specimen than routine culture (1.34 vs. 0.47 per specimen). Independent verification using 16S rRNA sequencing and culture (n = 10) corroborated 58% of additional detections compared to routine microbiology. Overall the average sensitivity for organism detection by Unyvero P55 was 88.8% and specificity was 94.9%. While Unyvero P55 detected more antimicrobial resistance markers than routine culture, some instances of phenotypic resistance were missed.

Conclusions

The Unyvero P55 is a rapid pathogen detection test for lower respiratory specimens, which identifies a larger number of pathogens than routine microbiology. The clinical significance of these additional organisms is yet to be determined. Further studies are required to determine the effect of the test in practise on antimicrobial prescribing and patient outcomes.