Background and Objectives: Liver cirrhosis is a serious and chronic condition that affects the liver, causing irreversible damage and scarring. The present study is designed to find out a possible correlation of hepatitis B virus (HBV) with cirrhosis by microRNA.Methods: The fold expression of the identified microRNAs by RT qPCR was determined to estimate the concentrations of circulating microRNAs in all samples. This study's main objective was to examine microRNA21-5p expression in liver cirrhosis patients. To extract RNA from samples of whole blood from all specimens in EDTA tubes, the study entailed collecting 60 specimens from the perspective of patients and samples pooled from 60 healthy participants (control group). Data on the patient are gathered for the study.Result: Researchers compared the levels of miRNA-146a in individuals with hepatic cirrhosis and control subjects, and the findings were clear. individuals with hepatic cirrhoses and controls, correspondingly, had mean levels of miRNA-146a of 2.38 3.25 and 1.12 1.01; this alteration was statistically noteworthy (P = 0.002). Both the miRNA-146a cut-off value and the prediction of liver cirrhosis disease should be considered diagnostic or adjuvant diagnostic tests. With sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of 58.3%, 53.3%, 55.6%, 56.1%, and 0.610 (0.508 -0.711), the miRNA-146a cut-off value was > 0.91-fold. The current findings suggest that miRNA-146a is a subpar diagnostic marker.Conclusion: Compared to controls, patients with cirrhosis had significantly higher levels of micro-RNA146A. When compared to those who are healthy, this finding demonstrates that micro-RNA 146A may influence the prognosis of cirrhosis.
- MeSH
- biochemická analýza krve metody MeSH
- exprese genu MeSH
- hepatitida B diagnóza genetika MeSH
- jaterní cirhóza * diagnóza genetika MeSH
- klinická studie jako téma metody MeSH
- lidé MeSH
- mikro RNA * genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
Cíl: Endometriální polypy (EP) představují patologii, která je v klinické praxi běžná. Ačkoli jejich přesná etiologie není úplně známá, existují důkazy o tom, že jsou citlivé vůči hormonální stimulaci. Naším cílem bylo prozkoumat vztah mezi kisspeptinem (KP) a EP pomocí srovnání míry genetické exprese (tkáň–krev) a imunohistochemické (IHC) exprese KP v lézích EP u pacientek s normálním endometriálním nálezem. Materiál a metody: Byla provedena retrospektivní případová studie u 50 pacientek s EP (n = 25) a normálním endometriálním nálezem (n = 25) z biopsie a/nebo materiálu z excize. Krev a vzorky z biopsie od všech pacientek byly uchovávány při –80 °C. Exprese genů KP byla stanovena v parafinových blocích a vzorcích periferní žilní krve získaných z bioptických vzorků a analýza skóre IHC-H byla provedena na parafinových blocích. EP a příslušné kontroly byly srovnány z hlediska KP. Výsledky: Po IHC bylo KP-H skóre v kontrolní skupině vyšší než ve skupině s EP a tento rozdíl byl statisticky významný; H skóre: kontroly: 5 (++; 1–15); polypy: 1 (+; 0–12) (p < 0,05). Ačkoli byla exprese KP jak v tkáni, tak v krvi vyšší v kontrolní skupině oproti skupině s EP, tento rozdíl nebyl statisticky významný (p > 0,05). V krvi ani tkáni nebyla zjištěna významná korelace mezi IHC-H skóre a expresí KP. Podle analýzy ROC cut-off hodnoty exprese KP v tkáni a krvi a plocha pod křivkou (AUC), která predikuje pravděpodobnost vzniku EP, nebyly významné (KP v tkáni: 1,04; AUC: 0,570; p = 0,388; senzitivita 56 %, specificita 60 % / KP v krvi: 1,06; AUC: 0,569; p = 0,401; senzitivita 80 %, specificita 40 %). Závěry: Snížená míra exprese KP v lézích EP může predikovat diagnózu EP a v budoucnu může mít KP při benigních gynekologických patologiích, jako jsou polypy, terapeutický potenciál.
Objective: Endometrial polyp (EP) is a type of pathology that is quite common in clinical practice. Although its exact etiology is not fully known, there is evidence to support that it is sensitive to hormonal stimuli. We aimed to investigate the relationship between kisspeptin (KP) and EP by comparing the genetic (tissue-blood) and immunohistochemical (IHC) expression of KP in EP lesions in patients with normal endometrial findings. Materials and methods: A prospective case-control study of 50 patients with EP (N = 25) and normal endometrial findings (N = 25) on biopsy and/or excision material was performed. Blood and biopsy samples obtained from all patients were stored at –80 °C. KP gene expression levels were determined from paraffin blocks, and peripheral venous blood samples obtained from biopsy specimens and IHC-H-score analysis were performed from paraffin blocks. EP and matched controls were compared for KP. Results: After IHC, the KP H-score of the control group was higher than the EP group, and this difference was statistically significant; H-score: control: 5 (++; 1–15); polyp: 1 (+; 0–12) (P < 0.05). Although KP expression in both tissue and blood was higher in the control group than in the EP group, this difference was not statistically significant (P > 0.05). No significant correlation was found between IHC H-score and KP expression levels in tissue and blood. According to the ROC analysis, the tissue and blood KP expression cut-off value and area under the curve (AUC) predicting the likelihood of developing EP were not significant (tissue KP: 1.04, AUC: 0.570, P = 0.388, sensitivity 56%, specificity 60%, Blood KP: 1.06, AUC: 0.569, P = 0.401, sensitivity 80%, specificity 40%). Conclusions: Decreased KP expression level in EP lesions may predict the diagnosis of EP, and in the future, KP may have therapeutic potential for benign gynecological pathologies such as polyps.
- MeSH
- endometrium * metabolismus patologie MeSH
- imunohistochemie metody MeSH
- kisspeptiny genetika metabolismus MeSH
- lidé MeSH
- nemoci dělohy genetika krev metabolismus patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- polypy genetika metabolismus patologie MeSH
- prospektivní studie MeSH
- studie případů a kontrol MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- MeSH
- Bordetella patogenita MeSH
- klinické laboratorní techniky klasifikace metody MeSH
- lidé MeSH
- mikrobiologické techniky metody přístrojové vybavení MeSH
- pertuse * diagnóza etiologie mikrobiologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- sérologické testy metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- směrnice pro lékařskou praxi MeSH
Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.
- MeSH
- bcr-abl fúzní proteiny krev MeSH
- buňky K562 chemie MeSH
- chronická myeloidní leukemie krev MeSH
- HL-60 buňky chemie MeSH
- klinické laboratoře MeSH
- lidé MeSH
- lineární modely MeSH
- nádorové biomarkery krev MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- reagenční diagnostické soupravy MeSH
- reprodukovatelnost výsledků MeSH
- testování odbornosti laboratoří * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Asie MeSH
- Evropa MeSH
- Severní Amerika MeSH
Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.
- MeSH
- exprese genu MeSH
- hmyz - vektory parazitologie MeSH
- Leishmania infantum genetika MeSH
- Leishmania izolace a purifikace MeSH
- leishmanióza viscerální epidemiologie parazitologie přenos MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- Psychodidae genetika parazitologie MeSH
- stadia vývoje MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Brazílie MeSH
Introduction: Cancer stem cells (CSCs) are a theorized subset of cells within the tumor that is thought to drive disease recurrence and metastatic spread. The aim of this study is to investigate mRNA and protein levels of ganglioside GD2 synthase (GD2S), in breast cancer (BC) patients. Methods: 65 PBMCs of preoperative BC patients without chemotherapy were compared to PBMCs after chemotherapy and controls. Results: GD2S were significantly higher in BC patients after chemotherapy compared to pre-chemotherapy at both mRNA and protein. GD2S was higher in pre-chemotherapy blood samples compared to control samples. Conclusions: Higher expression of GD2S in BC samples compared to healthy control indicates the potential utility of GD2S as a marker of malignancy.
- MeSH
- ELISA metody MeSH
- lidé MeSH
- lokální recidiva nádoru MeSH
- messenger RNA metabolismus MeSH
- N-acetylgalaktosaminyltransferasy krev MeSH
- nádorové kmenové buňky enzymologie metabolismus MeSH
- nádory prsu diagnóza farmakoterapie genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.
- MeSH
- chřipka lidská diagnóza virologie MeSH
- infekce viry z čeledi Orthomyxoviridae diagnóza virologie MeSH
- lidé MeSH
- nemoci prasat diagnóza virologie MeSH
- One Health MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- prasata MeSH
- ptačí chřipka u ptáků diagnóza virologie MeSH
- ptáci virologie MeSH
- reprodukovatelnost výsledků MeSH
- virus chřipky A, podtyp H1N1 genetika izolace a purifikace MeSH
- virus chřipky A, podtyp H3N2 genetika izolace a purifikace MeSH
- virus chřipky A genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
BACKGROUND: Recent advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on reverse transcription (RT). However, RT is recognized as a known source of variability, particularly with low amounts of RNA. Recently, several new reverse transcriptases (RTases) with the potential to decrease the loss of information have been developed, but knowledge of their performance is limited. METHODS: We compared the performance of 11 RTases in quantitative reverse transcription PCR (RT-qPCR) on single-cell and 100-cell bulk templates, using 2 priming strategies: a conventional mixture of random hexamers with oligo(dT)s and a reduced concentration of oligo(dT)s mimicking common single-cell RNA-sequencing protocols. Depending on their performance, 2 RTases were further tested in a high-throughput single-cell experiment. RESULTS: All tested RTases demonstrated high precision (R2 > 0.9445). The most pronounced differences were found in their ability to capture rare transcripts (0%-90% reaction positivity rate) and in their absolute reaction yield (7.3%-137.9%). RTase performance and reproducibility were compared with Z scores. The 2 best-performing enzymes were Maxima H- and SuperScript IV. The validity of the obtained results was confirmed in a follow-up single-cell model experiment. The better-performing enzyme (Maxima H-) increased the sensitivity of the single-cell experiment and improved resolution in the clustering analysis over the commonly used RTase (SuperScript II). CONCLUSIONS: Our comprehensive comparison of 11 RTases in low RNA input conditions identified 2 best-performing enzymes. Our results provide a point of reference for the improvement of current single-cell quantification protocols.
- MeSH
- analýza jednotlivých buněk MeSH
- DNA primery metabolismus MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- reprodukovatelnost výsledků MeSH
- reverzní transkriptasa metabolismus MeSH
- RNA metabolismus MeSH
- superoxiddismutasa 1 genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.
- MeSH
- Caenorhabditis elegans chemie MeSH
- chemická frakcionace metody MeSH
- cirkulující mikroRNA krev izolace a purifikace MeSH
- extracelulární vezikuly chemie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery krev izolace a purifikace MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- senioři MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.
