Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference.

Analysis of microRNAs in Small Urinary Extracellular Vesicles and Their Potential Roles in Pathogenesis of Renal ANCA-Associated Vasculitis.

Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) represents an autoimmunity disease characterized by high mortality. For successful treatment, the detailed knowledge of its complex pathogenesis and the set of biomarkers for differential diagnostics are desired. Analysis of molecular content of small urinary extracellular vesicles (uEV) offers the possibility to find markers in the form of microRNAs (miRNAs) and study the pathways involved in pathogenesis.

Mutational landscape of plasma cell-free DNA identifies molecular features associated with therapeutic response in patients with colon cancer. A pilot study.

Cell-free DNA (cfDNA) has recently been used as a non-invasive diagnostic tool for detecting tumour-specific mutations. cfDNA may also be used for monitoring disease progression and treatment response, but so far researchers focused on one or few genes only. A genomic profile may provide better information on patient prognosis compared to single specific mutations.

Optimization of diagnostic strategy for non-invasive cell-free foetal RHD determination from maternal plasma.

Background And Objectives: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis.

Materials And Methods: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma.

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR.

In families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals.

Comparison of MicroRNA Content in Plasma and Urine Indicates the Existence of a Transrenal Passage of Selected MicroRNAs.

MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples from 70 individuals (45 males, 25 females, mean age 65 years, range 20-84 years). Of the urine donors, 15 were healthy volunteers, 5 were patients with non-cancer diseases, 50 were patients with different stages of bladder cancer.

Differentially expressed miRNAs in trisomy 21 placentas.

Objective: Molecular pathogenesis of Down syndrome (DS) is still incompletely understood. Epigenetic mechanisms, including miRNAs gene expression regulation, belong to potential influencing factors. The aims of this study were to compare miRNAs expressions in placentas with normal and trisomic karyotype and to associate differentially expressed miRNAs with concrete biological pathways.

Serum microRNA-196 and microRNA-200 in pancreatic ductal adenocarcinoma of patients with diabetes mellitus.

Background/objectives: Our aim was to compare expressions of 6 microRNAs (miRNAs) in patients with pancreatic ductal adenocarcinoma (PAC) and non-cancer patients, moreover according to the presence or absence of diabetes mellitus.

Methods: Expressions of miRNA-192, -196, -200, -21, -30 and -423 were measured in 77 patients with PAC and 64 non-cancer patients (34 patients with type 2 DM and 30 control persons). 60 patients with PAC (78%) had DM or prediabetes and it was of new-onset (less than 2 years before the cancer diagnosis) in 44 out of them.

Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach.

Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used.

The impact of standard chemotherapy on miRNA signature in plasma in AML patients.

Aim: In our pilot study, we used plasma samples as liquid biopsy to search for miRNA signatures in patients with acute myeloid leukemia (AML) at diagnosis and in remission achieved after standard chemotherapy before planned transplantation.

Material And Methods: We examined 10 plasma samples from healthy volunteers and 8 paired samples from patients with AML at diagnosis and in remission using TaqMan MicroRNA Arrays. The results were validated using single-target qPCR reactions run in triplicates.

Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer.

Introduction: Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker.

Material And Methods: In total, 66 patients and 34 controls were enrolled into the study.

Quantification of circulating fetal DNA as a tool for potential monitoring of pregnant patients with antiphospholipid antibodies.

Apoptosis of tissues of fetal origin is thought to be one of the main sources of cell-free fetal DNA (cffDNA) in maternal circulation, impaired apoptosis is also involved in the mechanisms contributing to recurrent spontaneous miscarriages (RSM) associated with antiphospholipid syndrome (APS). The APS increases the risk for preeclampsia nine times. In preeclampsia, the elevated levels of cffDNA were described by different authors.

Methylation status of immune response genes promoters in cell-free DNA differs in hemodialyzed patients with diabetic nephropathy according to the intensity of anemia therapy.

Background: Anemia is a major complication of end-stage renal disease. Hemodialysis itself is regarded as a stimulus activating inflammation. Pro-inflammatory cytokines are able to suppress erythropoiesis.

Cell-free nucleic acids as biomarkers in dialyzed patients.

In this review, we discuss the origin, possible biological meaning, quantitative and qualitative changes in the concentrations of cell-free nucleic acids in human circulation with regard to renal failure and the process of dialysis. We focus on the inflammatory response and apoptosis known to be in close relationship not only with hemodialysis but also with different comorbidities frequently detected in hemodialyzed patients. 
Hemodialysis itself is able to promote the changes in the quantity and quality of circulating nucleic acid pool, but large spectrum of comorbidities in hemodialyzed subjects can further complicate the interpretations of results of cell-free nucleic acid analysis.

Alterations in methylation status of immune response genes promoters in cell-free DNA during a hemodialysis procedure.

Objective: Elevations of cell-free DNA (cfDNA) concentrations during hemodialysis (HD) sessions were reported in numerous studies regardless of an applied therapeutic protocol. It is generally thought that the elevated concentrations represent the consequence of apoptosis on the dialysis membranes. No data concerning the qualitative characteristics of cfDNAs in HD patients have been published till today; therefore, we focus on the promoter methylation status of genes involved in immune response.