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PubMed2026

Towards a Robust cell-free DNA Isolation Protocol for NGS Applications in a Clinical Molecular Diagnostics Setting.

Cell-free DNA (cfDNA), released from apoptotic and necrotic cells into body fluids, is a non-invasive source of genetic information for disease prediction, diagnosis, and monitoring. However, its low abundance makes cfDNA highly susceptible to various pre-analytical influences, potentially increasing high molecular weight (HMW) or genomic DNA (gDNA) compromising downstream cfDNA analyses. This study evaluated the impact of different cfDNA-stabilizing blood collection tubes (BCT; Cell-Free DNA BCT, Streck; S-Monovette cfDNA Exact, Sarstedt) stored at room temperature for 1, 5, or 10 days, prior to plasma isolation using different isolation methods (magnetic bead-based or silica column-based) on cfDNA stability and yield. DNA quantity and quality were assessed by fluorometric quantification, automated fragment analysis, and gene-specific quantitative PCR. Streck-based workflows maintained stable cfDNA yields and characteristic mononucleosomal fragmentation profiles across all storage times. In contrast, Sarstedt tubes showed reduced cfDNA concentrations after 5 days and a pronounced increase at 10 Days, accompanied by high-molecular weight DNA patterns consistent with white-blood cells (WBC) lysis. These trends were largely independent of the extraction method. Overall, the results demonstrate that blood collection tube chemistry critically influences cfDNA integrity during delayed processing. Streck tubes, particularly when combined with silica column-based isolation method, provided the most robust and reproducible workflow for routine molecular diagnostics, whereas Sarstedt tubes produced physiologically implausible results after extended storage.

blood collection tubes↗

PubMed2026

A species-discriminatory aerA TaqMan qPCR assay for rapid quantification of Aeromonas veronii in fish tissues and aquaculture water.

Aeromonas veronii is a major bacterial pathogen in freshwater aquaculture, yet rapid species-level quantification remains challenging within the genetically complex genus Aeromonas. We developed a singleplex hydrolysis-probe (TaqMan) quantitative PCR (qPCR) assay targeting an A. veronii-discriminatory region of the aerolysin gene (aerA) and validated it according to MIQE recommendations. Plasmid standards gave a linear range of 2 to 2 × 106 copies/reaction (R2 = 0.9962) with 100.5% amplification efficiency. The endpoint limit of detection was 2 copies per reaction, and 20 copies per reaction was set as the practical reporting limit based on reproducible detection and low intra- and inter-assay variation. Analytical specificity was evaluated with genomic DNA from an 18-strain panel, with reproducible amplification observed only for A. veronii. The assay was further tested in 55 fish-tissue and 11 aquaculture-water DNA extracts. NH8B-1D2 sample-process monitoring was used for matrix-level recovery correction, and tissue and water extraction blanks were undetermined. The aerA target was detected in all tested gill, stomach/intestine, spleen, kidney/head kidney, pond-water filter and Xiamen seawater filter extracts, and in 10/11 liver extracts. Median NH8B-corrected loads were highest in gill among tissues and higher in pond-water filters than in Xiamen seawater filters. A separate Vibrio harveyi inhibition-check assay indicated no obvious amplification-stage inhibition. This assay supports rapid quantification of aerA-positive A. veronii in fish and aquaculture-water matrices.

Animals↗

PubMed2003

URP1: a member of a novel family of PH and FERM domain-containing membrane-associated proteins is significantly over-expressed in lung and colon carcinomas.

In a concerted effort to identify biomarkers for lung and colon carcinomas by genome-wide transcriptional profiling, we describe the identification and cloning of one such gene as well as two additional closely related genes. Due to the strong sequence homology to the C. elegans UNC-112 we call this gene URP1, for UNC-112 related protein. We have also isolated the full-length clones for another novel related gene, URP2 and the previously discovered MIG-2 gene. Collectively, these proteins, together with two from Drosophila, appear to form a novel membrane-associated FERM and PH domain-containing protein family. Transcriptional analysis shows that only URP1 is significantly differentially regulated, being over-expressed in 70% of the colon carcinomas and 60% of the lung carcinomas tested. Quantification of URP1 expression by qRT-PCR showed up-regulation of the gene by 60-fold in lung tumors and up to nearly 6-fold in colon tumors. Northern blot analysis of URP1 indicates that normal expression is restricted to neuromuscular tissues. In contrast, the expression of URP2 appears to be confined primarily to tissues of the immune system. SNP analysis of URP1 reveals that it is highly polymorphic, containing seven sites, four of which are in the coding region and one position that results in the interchangeable substitution of glutamic acid and lysine. Finally, we have shown that the genomic structure for all three genes is nearly identical with all encoded by 15 exons although URP1 gene localized to chromosome 20p13, URP2 to 11q12 and MIG-2 to 14q22. This conserved exon structure suggests that all three members probably arose by gene duplication from one ancestral gene. The presence of multiple FERM domains characteristic of cytoplasmic plasma membrane to cytoskeleton linkers and a PH domain typical of membrane-anchored proteins involved in signal transduction suggest an important role for URP1 in tumorigenesis.

Amino Acid Sequence↗

PubMed1992

Identification and quantification of rodent malaria strains and species using gene probes.

A DNA probe PCsv4 and a subclone thereof PCsv4.1, hybridize specifically to rodent malaria DNA. DNA purified from a small volume (10 microliters) of infected mouse blood was used to determine the composition of the parasite population present. The hybridization signal following PCsv4 probing of slot-blotted DNA correlated directly with parasitaemia. The hybridization pattern and intensity, resulting from probing restriction enzyme digested and Southern-blotted genomic DNA, determined the identity of the infecting parasite line(s), and provided a semi-quantitative measure of parasite burden. Fifteen parasite lines representative of all four Plasmodium species infecting rodents can be differentiated in this way.

Animals↗

PubMed1996

Quantitative competitive polymerase chain reaction: analysis of amplified products of the HIV-1 gag gene by capillary electrophoresis with laser-induced fluorescence detection.

A quantitative competitive polymerase chain reaction (PCR) assay for the detection and quantification of HIV-1 has been developed using capillary electrophoresis. Separation of the PCR products is carried out in coated capillaries filled with replaceable linear polyacrylamide, and detection is performed using laser-induced fluorescence. The quantitative capabilities of this assay are described. We show results from analysis of a noninfectious plasmid encoding the HIV genome and integrated proviral DNA. Potential applications of this assay are discussed.

Cell Line↗

PubMed2006

[DNA sperm methylation in assisted reproductive techniques].

In the last few years, many tests were developed to study the fertilizing properties of the spermatozoa. However none of them was useful to obtain a prognostic factor. Indeed, the integrity of the spermatic DNA is also necessary to a successful fertilization for obtaining a pregnancy. DNA integrity could be evaluated by the measurement of the level of DNA methylation. Indeed, in the mammals, the methylation of the ADN is involved in diverse processes amongst them the regulation of the genome expression during the embryonic development. The objective of this study is to evaluate the impact of the level of methylation of the spermatic DNA in the success of in vitro fertilization (IVF), in terms of rate of fertilization, quality of the embryos and rate of pregnancy. The immunostaining of the 5-methylecytosine, then the quantification by image analysis or with flow cytometry, allowed an objective evaluation of the level of total methylation of spermatic DNA. Our data show that the level of DNA methylation influences neither the fertilization rate nor the embryos quality. On the other hand, the rate of pregnancy is decreased if the total level of DNA methylation is lower than a threshold value. The level of spermatic DNA methylation represents a new parameter of spermatic maturation.

5-Methylcytosine↗

PubMed2005

Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat.

Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.

Base Sequence↗

PubMed2006

Authentication of coffee by means of PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis.

Coffee is one of the most important world food commodities, commercial trade consisting almost entirely of Arabica and Robusta varieties. The former is considered to be of superior quality and thus attracts a premium price. Methods to differentiate these coffee species could prove to be beneficial for the detection of either deliberate or accidental adulteration. This study describes a molecular genetics approach to differentiate Arabica and Robusta coffee beans. This employs a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism to monitor a single nucleotide polymorphism within the chloroplastic genome. Samples were analyzed with a lab-on-a-chip capillary electrophoresis system. Coffee powder mixtures were analyzed with this technique, displaying a 5% limit of detection. The plastid copy number was found to be relatively constant across a wide range of bean samples, suggesting that this methodology can also be employed for the quantification of any adulteration of Arabica with Robusta beans.

Chloroplasts↗

PubMed2005

Highly consistent patterns for inherited human diseases at the molecular level.

Over 1600 mammalian genes are known to cause an inherited disorder, when subjected to one or more mutations. These disease genes represent a unique resource for the identification and quantification of relationships between phenotypic attributes of a disease and the molecular features of the associated disease genes, including their ascribed annotated functional classes and expression patterns. Such analyses can provide a more global perspective and a deeper understanding of the probable causes underlying human hereditary diseases. In this perspective and critical view of disease genomics, we present a comparative analysis of genes reported to cause inherited diseases in humans in terms of their causative effects on physiology, their genetics and inheritance modes, the functional processes they are involved in and their expression profiles across a wide spectrum of tissues. Our analysis reveals that there are more extensive correlations between these attributes of genetic disease genes than previously appreciated. For instance, the functional pattern of genes causing dominant and recessive diseases is markedly different. Also, the function of the genes and their expression correlate with the type of disease they cause when mutated. The results further indicate that a comparative genomics approach for the analysis of genes linked to human genetic diseases will facilitate the elucidation of the underlying molecular and cellular mechanisms.

Biomarkers↗

PubMed2000

Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples.

A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5'-3' nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997-1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998-1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997-1998 and 1998-1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.

DNA Primers↗

PubMed1997

Sensitive chemiluminescence in situ hybridization for the detection of human papillomavirus genomes in biopsy specimens.

We developed a sensitive chemiluminescence in situ hybridization assay for detection of human papillomavirus (HPV) DNA for objective and semiquantitative evaluation of the results. The hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes, visualized with alkaline phosphatase as the revealing enzyme and a highly sensitive 1,2 dioxetane phosphate as chemiluminescent substrate. The light emitted from the hybridized probes was detected, analyzed, and measured using a high-performance, low light-level imaging luminograph connected to an optical microscope and to a personal computer for quantification of the photon fluxes and for image analysis. The system operated in consecutive steps: First, hybridized specimens were recorded in transmitted light. Then the net luminescent signal was recorded, and then an overlay of the two images provided by the transmitted light and by the luminescent signal allowed the spatial distribution of the target DNA to be localized, measured, and evaluated. Biopsy specimens from different pathological conditions associated with HPV, which had previously been proved positive for HPV DNA with the polymerase chain reaction (PCR), were analysed. The chemiluminescence in situ hybridization proved sensitive and specific with digoxigenin-, biotin-, or fluorescein-labeled probes, and provided an objective evaluation of the results. The results obtained with chemiluminescence in situ hybridization were also compared with results obtained with in situ hybridization with colorimetric detection, with good concordance of the data. Chemiluminescence in situ hybridization therefore offers the possibility of detecting HPV DNA with great sensitivity in biopsy specimens. Moreover, the images of the samples, stored in the computer, are a permanent record of the reaction and can also be sent for evaluation or comparison to other laboratories using computer networks.

Biotin↗

PubMed2007

Impact of in vitro production techniques on the expression of X-linked genes in bovine (bos taurus) oocytes and pre-attachment embryos.

Our previous studies showed that expression patterns of X-linked genes in cultured cells are different from those of their tissues of origin. This investigation analyses the transcription pattern of the X-linked genes BIRC4, GAB3, MECP2, RPS4X, SLC25A6, and XIST in bovine in vitro matured oocytes and in vitro fertilized embryos, and their in vivo counterparts. In vitro-derived pools of mature oocytes and pre-attachment embryos were obtained by: (a) TCM-199/serum with bovine oviductal epithelial cells as co-culture, and (b) synthetic oviductal fluid/BSA. Pools of in vivo-derived morulae and blastocysts were provided by a commercial embryo transfer operation. Total RNA was extracted for quantification of gene-specific transcript levels using real-time quantitative PCR. Statistical analysis was performed using a mixed model factorial ANOVA with alpha = 0.05. The effect of the in vitro environmental conditions on X-linked gene transcription was most evident during the fourth cell cycle, at the period of activation of the embryonic genome, and seemed to be less pronounced at later developmental stages, with the exception of BIRC4. The levels of X-linked genes transcripts in in vivo-derived embryos were lower relative to their in vitro counterparts for all genes tested. Finally, the pattern of expression of XIST in bovine oocytes and embryos was similar to that reported in humans. These results highlight the possibility that X-linked gene expression analysis is a useful tool to monitor the impact of reproductive biotechnologies on the developmental potential of embryos and aid in their improvement.

Animals↗

PubMed2006

The annexins of Dictyostelium.

Annexins are a highly conserved ubiquitous family of Ca2+- and phospholipid-binding proteins present in nearly all eukaryotic cells. Analysis of the Dictyostelium genome revealed the presence of two annexin genes, the annexin C1 gene (nxnA) giving rise to two isoforms of 47 and 51 kDa (previously synexin), and the annexin C2 gene (nxnB) coding for a 56-kDa protein with 33% sequence identity to annexin C1. Annexin C2 is expressed at very low and constant levels throughout development. Quantification by real-time PCR indicated that it is present in about 35-fold lower amounts compared to annexin C1. We have used a GFP-tagged annexin C2 to study its cellular distribution and dynamics. In cell fractionation studies, annexin C2 cofractionates with annexin C1 and is enriched in the 100,000 g pellet. Like annexin C1, GFP-AnxC2 stains the plasma membrane. In addition it is present in the perinuclear region and overlaps to some degree with the Golgi apparatus, whereas annexin C1 is present on intracellular membranes resembling endosomal membranes and in the nucleus. Annexin C2 is not observed in the nucleus. An annexin C1 mutant (SYN-) which shows a defect during multicellular development can be rescued by full-length annexin C1, whereas overexpression of GFP-AnxC2 did not rescue the developmental defect The data support the concept that annexins, although having a highly conserved structure, participate in different functions in a cell.

Amino Acid Sequence↗

PubMed2005

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR.

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

Analysis of Variance↗

PubMed2006

A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Cell motility is a complex biological process, involved in development, inflammation, homeostasis, and pathological processes such as the invasion and metastatic spread of cancer. Here, we describe a genomic screen designed to identify inhibitors of cell migration. A library of 10,996 small interfering RNAs (targeting 5,234 human genes) was screened for their ability to block the migration of a highly motile ovarian carcinoma cell line, SKOV-3, by using a 384-well wound-healing assay coupled with automated microscopy and wound quantification. Two or more small interfering RNAs against four genes, CDK7, DYRK1B, MAP4K4 (NIK/HGK) (MAP4K4, mitogen-activated protein 4 kinase 4), and SCCA-1 (SerpinB3), potently blocked the migration of SKOV-3 cells, concordant with reduced transcript levels. Further studies of the promigratory role of MAP4K4 showed that the knockdown of this transcript inhibited the migration of multiple carcinoma cell lines, indicating a broad role in cell motility and potently suppressed the invasion of SKOV-3 cells in vitro. The effect of MAP4K4 on cellular migration was found to be mediated through c-Jun N-terminal kinase, independent of AP1 activation and downstream transcription. Accordingly, small molecule inhibition of c-Jun N-terminal kinase suppressed SKOV-3 cell migration, underscoring the potential therapeutic utility of mitogen-activated protein kinase pathway inhibition in cancer progression.

Base Sequence↗

PubMed2025

StrainR2 accurately deconvolutes strain-level abundances in synthetic microbial communities.

MOTIVATION: Synthetic microbial communities offer an opportunity to conduct reductionist research in tractable model systems. However, deriving abundances of highly related strains within these communities is currently unreliable. 16S rRNA gene sequencing does not resolve abundance at the strain level and other methods such as quantitative polymerase chain reaction (qPCR) scale poorly and are resource prohibitive for complex communities. We present StrainR2, which utilizes shotgun metagenomic sequencing to provide high accuracy strain-level abundances for all members of a synthetic community, provided their genomes. RESULTS: Both in silico, and using sequencing data derived from gnotobiotic mice colonized with a synthetic fecal microbiota, StrainR2 resolves strain abundances with greater accuracy and efficiency than other tools utilizing shotgun metagenomic sequencing reads. We demonstrate that StrainR2's accuracy is comparable to that of qPCR on a subset of strains resolved using absolute quantification. AVAILABILITY AND IMPLEMENTATION: Software is available at GitHub and implemented in C, R, and Bash. Software is supported on Linux and MacOS, with packages available on Bioconda or as a Docker container. The source code at the time of publication is also available on figshare at the doi: 10.6084/m9.figshare.29420780.

Mice↗

PubMed2002

Fully automatic quantification of microarray image data.

DNA microarrays are now widely used to measure expression levels and DNA copy number in biological samples. Ratios of relative abundance of nucleic acids are derived from images of regular arrays of spots containing target genetic material to which fluorescently labeled samples are hybridized. Whereas there are a number of methods in use for the quantification of images, many of the software systems in wide use either encourage or require extensive human interaction at the level of individual spots on arrays. We present a fully automatic system for microarray image quantification. The system automatically locates both subarray grids and individual spots, requiring no user identification of any image coordinates. Ratios are computed based on explicit segmentation of each spot. On a typical image of 6000 spots, the entire process takes less than 20 sec. We present a quantitative assessment of performance on multiple replicates of genome-wide array-based comparative genomic hybridization experiments. By explicitly identifying the pixels in each spot, the system yields more accurate estimates of ratios than systems assuming spot circularity. The software, called, runs on Windows platforms and is available free of charge for academic use.

Breast Neoplasms↗

PubMed2004

Ovine dendritic cells transduced with an adenoviral CTLA4eEGFP fusion protein construct induce hyporesponsiveness to allostimulation.

CTLA4 (CD152) is a transmembrane molecule expressed on activated T cells and functions as a negative regulator of T cell activation upon binding to the costimulatory molecules CD80/86. In this study, CTLA4eEGFP constructs were engineered by cloning the extracellular domains of ovine and human CTLA4 (CTLA4e) 'in frame' with the enhanced green fluorescent protein (EGFP). Recombinant adenoviral vectors were generated by incorporation of the CTLA4eEGFP sequence into the adenoviral genome using homologous recombination in Esherichia coli. The functional activity of the adenoviral vectors was shown by the secretion of the CTLA4eEGFP upon infection of ovine fibroblasts and the binding of the fusion protein to the target ovine and human dendritic cells expressing CD80/86 receptors by flow cytometry. The EGFP tag facilitated molecular size determinations and quantification of the secreted ovine CTLA4 fusion protein by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA), respectively, using anti-GFP mAbs. Ovine dendritic cells obtained from pseudoafferent lymphatic cannulation of sheep were characterized based on high major histocompatibility complex (MHC) class II expression and cross-reactivity with monoclonal antibodies to the human dendritic cell markers, CD83 and CMRF-56. In addition, ovine dendritic cells (DC) were transfected with the adenoviral CTLA4eEGFP and when used as stimulators in a mixed lymphocyte reaction showed a reduced capacity to induce allogeneic lymphocyte proliferation. This study verifies that the ovine CTLA4eEGFP fusion protein functions similarly to its human homologue and that DC modified with adenoviral CTLA4-EGFP may provide an effective therapeutic approach in targeting alloreactive T cells to prolong allograft acceptance in a preclinical ovine model of renal transplantation.

Adenoviridae↗