Amount Of RNA Research Articles

The rise of single-cell transcriptomics in yeast.

The field of single-cell omics has transformed our understanding of biological processes and is constantly advancing both experimentally and computationally. One of the most significant developments is the ability to measure the transcriptome of individual cells by single-cell RNA-seq (scRNA-seq), which was pioneered in higher eukaryotes. While yeast has served as a powerful model organism in which to test and develop transcriptomic technologies, the implementation of scRNA-seq has been significantly delayed in this organism, mainly because of technical constraints associated with its intrinsic characteristics, namely the presence of a cell wall, a small cell size and little amounts of RNA. In this review, we examine the current technologies for scRNA-seq in yeast and highlight their strengths and weaknesses. Additionally, we explore opportunities for developing novel technologies and the potential outcomes of implementing single-cell transcriptomics and extension to other modalities. Undoubtedly, scRNA-seq will be invaluable for both basic and applied yeast research, providing unique insights into fundamental biological processes.

Laboratory-based evaluation of the 4th-generation AlereTM HIV Combo rapid point-of-care test.

Mozambique is a high-prevalence country for HIV and early detection of new HIV infections is crucial for control of the epidemic. We aimed to evaluate the accuracy of the 4th-generation rapid diagnostic test (RDT) AlereTM HIV Combo in detecting acute and seroconverted HIV-infection, among sexually-active women attending three clinical health centers in Maputo, Mozambique. Women aged 14-55 years (n = 920) seeking care at the Mavalane Health Area, Maputo (February 2018-January 2019) were included, and blood specimens sampled. Sociodemographic and sexual behavior data were collected. Point-of-care HIV testing was performed using Alere DetermineTM HIV-1/2 and Uni-GoldTM HIV-1/2. All samples were also tested using Enzygnost® HIV Integral 4 and Innotest® HIV Antigen mAb in laboratory. The 4th-generation RDT AlereTM HIV Combo was evaluated on serum samples in the laboratory. Finally, Innotest® HIV Antigen mAb, Enzygnost® HIV Integral 4 (Ag/Ab), and HIV RNA quantification acted as gold standard assays in the evaluation of AlereTM HIV Combo test for HIV antigen detection (in clinical samples and in three HIV-1 seroconversion panels). The antibody component of the 4th generation AlereTM HIV Combo RDT demonstrated a sensitivity and specificity of 100% examining clinical samples. However, the test did not detect HIV p24 antigen in any clinical samples, while Innotest® HIV Antigen mAb, verified by Enzygnost® HIV Integral 4 (Ag/Ab) and/or HIV RNA quantification, detected HIV antigen in six clinical samples. Furthermore, the AlereTM HIV Combo RDT had a low sensitivity in the detection of HIV p24 antigen in seroconversion panels. The HIV prevalence among the examined women was 17.8%. The 4th-generation RDT AlereTM HIV Combo showed similar sensitivity to the 3rd-generation RDTs to detect seroconverted HIV-infections. However, the sensitivity for detection of HIV p24 antigen and diagnosing acute HIV infections, before seroconversion, was low. There is an urgent need to develop and evaluate simple and affordable POC tests with high sensitivity and specificity for diagnosing individuals with acute HIV infection in resource-limited settings with high HIV prevalence.

An iron rheostat controls hematopoietic stem cell fate

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.

Low unspliced cell-associated HIV RNA in early treated adolescents living with HIV on long suppressive ART.

Initiation of antiretroviral treatment (ART) in patients early after HIV-infection and long-term suppression leads to low or undetectable levels of HIV RNA and cell-associated (CA) HIV DNA and RNA. Both CA-DNA and CA-RNA, overestimate the size of the HIV reservoir but CA-RNA as well as p24/cell-free viral RNA can be indicators of residual viral replication. This study describes HIV RNA amounts and levels of cytokines/soluble markers in 40 well-suppressed adolescents who initiated ART early in life and investigated which viral markers may be informative as endpoints in cure clinical trials within this population. Forty adolescents perinatally infected with HIV on suppressive ART for >5 years were enrolled in the CARMA study. HIV DNA and total or unspliced CA-RNA in PBMCs were analyzed by qPCR/RT-qPCR and dPCR/RT-dPCR. Cell-free HIV was determined using an ultrasensitive viral load (US-VL) assay. Plasma markers and p24 were analyzed by digital ELISA and correlations between total and unspliced HIV RNA and clinical markers, including age at ART, Western Blot score, levels of cytokines/inflammation markers or HIV CA-DNA, were tested. CA-RNA was detected in two thirds of the participants and was comparable in RT-qPCR and RT-dPCR. Adolescents with undetectable CA-RNA showed significantly lower HIV DNA compared to individuals with detectable CA-RNA. Undetectable unspliced CA-RNA was positively associated with age at ART initiation and Western Blot score. We found that a higher concentration of TNF-α was predictive of higher CA-DNA and CA-RNA. Other clinical characteristics like US-VL, time to suppression, or percent CD4+ T-lymphocytes were not predictive of the CA-RNA in this cross-sectional study. Low CA-DNA after long-term suppressive ART is associated with lower CA-RNA, in concordance with other reports. Patients with low CA-RNA levels in combination with low CA-DNA and low Western Blot scores should be further investigated to characterize candidates for treatment interruption trials. Unspliced CA-RNA warrants further investigation as a marker that can be prioritized in paediatric clinical trials where the sample volume can be a significant limitation.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at − 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Small RNAs: an ideal choice as sperm quality biomarkers.

Spermatozoa were classically known as vehicles for the delivery of the paternal genome to the oocyte. However, in 1962, spermatozoa were discovered to carry significant amounts of RNA in them, which raised questions about the significance of these molecules in such a highly specialized cell. Scientific research in the last six decades has investigated the biological significance of sperm RNAs by various means. Irrespective of what sperm RNAs do, their presence in spermatozoa has attracted attention for their exploitation as biomarkers of fertility. Research in this direction started in the year 2000 and is still underway. A major hurdle in this research is the definition of the standard human sperm RNAome. Only a few normozoospermic samples have been analyzed to define the normal sperm RNAome. In this article, we provide a perspective on the suitability of sperm RNAs as biomarkers of fertility and the importance of defining the normal sperm RNAome before we can succeed in identifying RNA-based biomarkers of sperm quality and fertility. The identification of sperm RNA biomarkers of fertility can be exploited for quality screening of donor sperm samples, explain infertility in idiopathic cases, and RNA therapeutics for the treatment of male infertility.

Trans Species RNA Activity: Sperm RNA of the Father of an Autistic Child Programs Glial Cells and Behavioral Disorders in Mice.

Recently, we described the alteration of six miRNAs in the serum of autistic children, their fathers, mothers, siblings, and in the sperm of autistic mouse models. Studies in model organisms suggest that noncoding RNAs participate in transcriptional modulation pathways. Using mice, approaches to alter the amount of RNA in fertilized eggs enable in vivo intervention at an early stage of development. Noncoding RNAs are very numerous in spermatozoa. Our study addresses a fundamental question: can the transfer of RNA content from sperm to eggs result in changes in phenotypic traits, such as autism? To explore this, we used sperm RNA from a normal father but with autistic children to create mouse models for autism. Here, we induced, in a single step by microinjecting sperm RNA into fertilized mouse eggs, a transcriptional alteration with the transformation in adults of glial cells into cells affected by astrogliosis and microgliosis developing deficiency disorders of the 'autism-like' type in mice born following these manipulations. Human sperm RNA alters gene expression in mice, and validates the possibility of non-Mendelian inheritance in autism.

Quantification of plasma HDV RNA in untreated and Bulevirtide-treated patients with CHD: a comparison between Robogene 2.0, Eurobioplex and Altostar

Quantification of plasma HDV RNA in untreated and Bulevirtide-treated patients with CHD: a comparison between Robogene 2.0, Eurobioplex and Altostar

ZNF148 inhibits HBV replication by downregulating RXRα transcription

BackgroundProgressive hepatitis B virus (HBV) infection can result in cirrhosis, hepatocellular cancer, and chronic hepatitis. While antiviral drugs that are now on the market are efficient in controlling HBV infection, finding a functional cure is still quite difficult. Identifying host factors involved in regulating the HBV life cycle will contribute to the development of new antiviral strategies. Zinc finger proteins have a significant function in HBV replication, according to earlier studies. Zinc finger protein 148 (ZNF148), a zinc finger transcription factor, regulates the expression of various genes by specifically binding to GC-rich sequences within promoter regions. The function of ZNF148 in HBV replication was investigated in this study.MethodsHepG2-Na+/taurocholate cotransporting polypeptide (HepG2-NTCP) cells and Huh7 cells were used to evaluate the function of ZNF148 in vitro. Northern blotting and real-time PCR were used to quantify the amount of viral RNA. Southern blotting and real-time PCR were used to quantify the amount of viral DNA. Viral protein levels were elevated, according to the Western blot results. Dual-luciferase reporter assays were used to examine the transcriptional activity of viral promoters. ZNF148’s impact on HBV in vivo was investigated using an established rcccDNA mouse model.ResultsZNF148 overexpression significantly decreased the levels of HBV RNAs and HBV core DNA in HBV-infected HepG2-NTCP cells and Huh7 cells expressing prcccDNA. Silencing ZNF148 exhibited the opposite effects in both cell lines. Furthermore, ZNF148 inhibited the activity of HBV ENII/Cp and the transcriptional activity of cccDNA. Mechanistic studies revealed that ZNF148 attenuated retinoid X receptor alpha (RXRα) expression by binding to the RXRα promoter sequence. RXRα binding site mutation or RXRα overexpression abolished the suppressive effect of ZNF148 on HBV replication. The inhibitory effect of ZNF148 was also observed in the rcccDNA mouse model.ConclusionsZNF148 inhibited HBV replication by downregulating RXRα transcription. Our findings reveal that ZNF148 may be a new target for anti-HBV strategies.

Characterization and Spike Gene Analysis of a Candidate Attenuated Live Bovine Coronavirus Vaccine

The bovine coronavirus (BCoV) KBR-1 strain, obtained from calf diarrhea samples collected in 2017, belongs to group GIIa. To attenuate this strain, it was subcultured continuously (up to 79 times) in HRT-18 cells, followed by 80–120 passages in MDBK cells. The KBR-1-p120 strain harvested from MDBK cells at passage 120 harbored 13 amino acid mutations in the spike gene. Additionally, the KBR-1-p120 strain showed a high viral titer and cytopathogenic effects in MDBK cells. Seven-day-old calves (negative for BCoV antigen and antibodies) that did not consume colostrum were orally inoculated with the attenuated candidate strain (KBR-1-p120), or with KBR-1 passaged 10 times (KBR-1-p10) in HRT-18 cells. Calves inoculated with KBR-1-p10 had a low diarrhea score, and BCoV RNA was detected at 3–7 days post-inoculation (DPI). The virus was also present in the duodenum, jejunum, and ileum at autopsy; however, calves inoculated with KBR-1-p120 had low levels of BCoV RNA in feces at 4–6 DPI, and no diarrhea. In addition, an extremely small amount of BCoV RNA was present in the jejunum and ileum at autopsy. The small intestines of calves inoculated with KBR-1-p120 were emulsified and used to infect calves two more times, but pathogenicity was not recovered. Therefore, the KBR-1-p120 strain has potential as a live vaccine candidate.

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules.

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated by denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 sec, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the reproducible, statistically significant reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of the poly(A) binding protein, PABPC1, with polyadenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, and cost-effective method to enable researchers to analyze expression, processing, binding, and decay of RNAs.

Quantification of serum HDV RNA by Robogene 2.0 in HDV patients is significantly influenced by the extraction methods.

Management of chronic hepatitis delta (CHD) requires reliable tests for HDV RNA quantification. The aim of the study was to compare two extraction methods for the quantification of HDV RNA in untreated and bulevirtide (BLV)-treated CHD patients. Frozen sera from untreated and BLV-treated CHD patients were tested in a single-centre study for HDV RNA levels (Robogene 2.0, Roboscreen GmbH, Leipzig, Germany; LOD 6 IU/mL) with two extraction methods: manual (INSTANT Virus RNA/DNA kit; Roboscreen GmbH, Leipzig, Germany) versus automated (EZ1 DSP Virus Kit; Qiagen, Hilden, Germany). BLV-treated patients were sampled at baseline and during therapy. Two hundred sixty-four sera collected from 157 CHD (139 untreated, 18 BLV-treated) patients were analysed: age 51 (28-78), 59% males, 90% of European origin, 60% cirrhotics, ALT 85 (17-889) U/L, HBsAg 3.8 (1.7-4.6) Log IU/mL, 81% HBV DNA undetectable, 98% HDV genotype 1. Median HDV RNA was 4.53 (.70-8.10) versus 3.77 (.70-6.93) Log IU/mL by manual versus automated extraction (p < .0001). Manual extraction reported similar HDV RNA levels in 31 (20%) patients, higher in 119 (76%) [+.5 and +1 log10 in 60; > +1 log10 in 59] and lower in 7 (4%). Among 18 BLV-treated patients, rates of HDV RNA < LOD significantly differed between the two assays at Weeks 16 and 24 (0% vs. 22%, p = .02; 11% vs. 44%, p = .03), but not at later timepoints. By contrast, virological response rates were similar. Quantification of HDV RNA by Robogene 2.0 is influenced by the extraction method, the manual extraction being 1 Log more sensitive.

Characterization of Variant RNAs Encapsidated during Bromovirus Infection by High-Throughput Sequencing.

Previously, we described the RNA recombinants accumulating in tissues infected with the bromoviruses BMV (Brome mosaic virus) and CCMV (Cowpea chlorotic mottle virus). In this work, we characterize the recombinants encapsidated inside the purified virion particles of BMV and CCMV. By using a tool called the Viral Recombination Mapper (ViReMa) that detects recombination junctions, we analyzed a high number of high-throughput sequencing (HTS) short RNA sequence reads. Over 28% of BMV or CCMV RNA reads did not perfectly map to the viral genomes. ViReMa identified 1.40% and 1.83% of these unmapped reads as the RNA recombinants, respectively, in BMV and CCMV. Intra-segmental crosses were more frequent than the inter-segmental ones. Most intra-segmental junctions carried short insertions/deletions (indels) and caused frameshift mutations. The mutation hotspots clustered mainly within the open reading frames. Substitutions of various lengths were also identified, whereas a small fraction of crosses occurred between viral and their host RNAs. Our data reveal that the virions can package detectable amounts of multivariate recombinant RNAs, contributing to the flexible nature of the viral genomes.

Evaluation of Truck Cab Decontamination Procedures following Inoculation with Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus.

This experiment aimed to evaluate commercially available disinfectants and their application methods against porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) on truck cab surfaces. Plastic, fabric, and rubber surfaces inoculated with PEDV or PRRSV were placed in a full-scale truck cab and then treated with one of eight randomly assigned disinfectant treatments. After application, surfaces were environmentally sampled with cotton gauze and tested for PEDV and PRRSV using qPCR duplex analysis. There was a disinfectant × surface interaction (p < 0.0001), indicating a detectable amount of PEDV or PRRSV RNA was impacted by disinfectant treatment and surface material. For rubber surfaces, 10% bleach application had lower detectable amounts of RNA compared to all other treatments (p < 0.05) except Intervention via misting fumigation, which was intermediate. In both fabric and plastic surfaces, there was no evidence (p > 0.05) of a difference in detectable RNA between disinfectant treatments. For disinfectant treatments, fabric surfaces with no chemical treatment had less detectable viral RNA compared to the corresponding plastic and rubber (p < 0.05). Intervention applied via pump sprayer to fabric surfaces had less detectable viral RNA than plastic (p < 0.05). Furthermore, 10% bleach applied via pump sprayer to fabric and rubber surfaces had less detectable viral RNA than plastic (p < 0.05). Also, a 10 h downtime, with no chemical application or gaseous fumigation for 10 h, applied to fabric surfaces had less detectable viral RNA than other surfaces (p < 0.05). Sixteen treatments were evaluated via swine bioassay, but all samples failed to produce infectivity. In summary, commercially available disinfectants successfully reduced detectable viral RNA on surfaces but did not eliminate viral genetic material, highlighting the importance of bioexclusion of pathogens of interest.

Understanding Spondyloarthritis Pathogenesis: The Promise of Single-Cell Profiling.

Single-cell profiling, either in suspension or within the tissue context, is a rapidly evolving field. The purpose of this review is to outline recent advancements and emerging trends with a specific focus on studies in spondyloarthritis. The introduction of sequencing-based approaches for the quantification of RNA, protein, or epigenetic modifications at single-cell resolution has provided a major boost to discovery-driven research. Fluorescent flow cytometry, mass cytometry, and image-based cytometry continue to evolve. Spatial transcriptomics and imaging mass cytometry have extended high-dimensional analysis to cells in tissues. Applications in spondyloarthritis include the indexing and functional characterization of cells, discovery of disease-associated cell states, and identification of signatures associated with therapeutic responses. Single-cell TCR-seq has provided evidence for clonal expansion of CD8+ T cells in spondyloarthritis. The use of single-cell profiling approaches in spondyloarthritis research is still in its early stages. Challenges include high cost and limited availability of diseased tissue samples. To harness the full potential of the rapidly expanding technical capabilities, large-scale collaborative efforts are imperative.

Ascites exosomal lncRNA PLADE enhances platinum sensitivity by inducing R-loops in ovarian cancer.

Cisplatin resistance is a major cause of therapeutic failure in patients with high-grade serous ovarian cancer (HGSOC). Long noncoding RNAs (lncRNAs) have emerged as key regulators of human cancers; however, their modes of action in HGSOC remain largely unknown. Here, we provide evidence to demonstrate that lncRNA Platinum sensitivity-related LncRNA from Ascites-Derived Exosomes (PLADE) transmitted by ascites exosomes enhance platinum sensitivity in HGSOC. PLADE exhibited significantly decreased expression in ascites exosomes and tumor tissues, as well as in the corresponding metastatic tumors from patients with HGSOC cisplatin-resistance. Moreover, HGSOC patients with higher PLADE expression levels exhibited longer progression-free survival. Gain- and loss-of-function studies have revealed that PLADE promotes cisplatin sensitivity by suppressing cell proliferation, migration and invasion, and enhancing apoptosis in vitro and in vivo. Furthermore, the functions of PLADE in increasing cisplatin sensitivity were proven to be transferred by exosomes to the cultured recipient cells and to the adjacent tumor tissues in mouse models. Mechanistically, PLADE binds to and downregulates heterogeneous nuclear ribonucleoprotein D (HNRNPD) by VHL-mediated ubiquitination, thus inducing an increased amount of RNA: DNA hybrids (R-loop) and DNA damage, consequently promoting cisplatin sensitivity in HGSOC. Collectively, these results shed light on the understanding of the vital roles of long noncoding RNAs in cancers.

Validation of genes for H-ARS severity prediction in leukemia patients - interspecies comparison, challenges, and promises

Purpose In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients. Material and Methods Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2–12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated. Results Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1–2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated. Conclusion Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.

An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Clinical Applications of Next-Generation Sequencing for Cancer Diagnostics and Targeted Cancer Therapy

Next-generation sequencing (NGS) is a revolutionary technology that has effectively sequence massive quantities of genomic DNA or RNA at a shorter time and a lower cost. The NGS is routinely used in oncology to tailor care for individual patient. In this review, we discuss the role of NGS how it can provide a more personalized approach to targeted cancer therapy. A literature review was conducted via PubMed and articles were screened for publication dates within the past 10 years. Primary literature consisting of clinical trials and review articles were utilized. Search terms included “(next-generation sequencing) AND (cancer)”, “(next-generation sequencing) AND (cancer diagnostics)”, and “(next-generation sequencing) AND “(cancer therapy)”.&#x0D; From the literature review, we found that NGS is indicated for daily practice as a diagnostic tool for advanced NSCLC, colon cancer, prostate cancer, cholangiocarcinoma, and some advanced rare cancers. We also found evidence suggesting that NGS is helpful in identifying actionable mutations that will, in turn, allow the patient to be matched to a more individualized cancer treatment.&#x0D; As oncology care continues to move in the direction of more personalized care, the potential applications of NGS in the field of oncology will continue to evolve. Our review will help further the understanding of NGS and provide context for its value in patient care and its ability to provide an avenue for targeted therapy.

Effects of genetic variation on the structure of RNA and protein.

Changes in the structure of RNA and protein, have an important impact on biological functions and are even important determinants of disease pathogenesis and treatment. Some genetic variations, including copy number variation, single nucleotide variation, and so on, can lead to changes in biological function and increased susceptibility to certain diseases by changing the structure of RNA or protein. With the development of structural biology and sequencing technology, a large amount of RNA and protein structure data and genetic variation data resources has emerged to be used to explain biological processes. Here, we reviewed the effects of genetic variation on the structure of RNAs and proteins, and investigated their impact on several diseases. An online resource (http://www.onethird-lab.com/gems/) to support convenient retrieval of common tools is also built. Finally, the challenges and future development of the effects of genetic variation on RNA and protein were discussed.