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Background Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. New methods Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. Results Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3–5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. Comparison with existing methods Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. Conclusion These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS.

During acute reperfusion, the expression profiles of long noncoding RNAs in adult rats with focal cerebral ischemia undergo broad changes. However, whether long noncoding RNAs are involved in neuroprotective effects following focal ischemic stroke in rats remains unclear. In this study, RNA isolation and library preparation was performed for long noncoding RNA sequencing, followed by determining the coding potential of identified long noncoding RNAs and target gene prediction. Differential expression analysis, long noncoding RNA functional enrichment analysis, and co-expression network analysis were performed comparing ischemic rats with and without ischemic postconditioning rats. Rats were subjected to ischemic postconditioning via the brief and repeated occlusion of the middle cerebral artery or femoral artery. Quantitative real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of differentially expressed long noncoding RNAs after ischemic postconditioning in a rat model of ischemic stroke. The results showed that ischemic postconditioning greatly affected the expression profile of long noncoding RNAs and mRNAs in the brains of rats that underwent ischemic stroke. The predicted target genes of some of the identified long noncoding RNAs (cis targets) were related to the cellular response to ischemia and stress, cytokine signal transduction, inflammation, and apoptosis signal transduction pathways. In addition, 15 significantly differentially expressed long noncoding RNAs were identified in the brains of rats subjected to ischemic postconditioning. Nine candidate long noncoding RNAs that may be related to ischemic postconditioning were identified by a long noncoding RNA expression profile and long noncoding RNA-mRNA co-expression network analysis. Expression levels were verified by quantitative real-time reverse transcription-polymerase chain reaction. These results suggested that the identified long noncoding RNAs may be involved in the neuroprotective effects associated with ischemic postconditioning following ischemic stroke. The experimental animal procedures were approved by the Animal Experiment Ethics Committee of Kunming Medical University (approval No. KMMU2018018) in January 2018.

This chapter focuses on the process of isolation of ribonucleic acid (RNA) from biological sources enriched in ribonucleases (RNase) and then protecting the RNA from degradation in the ensuing protocols. RNases are a ubiquitous family of enzymes, present in virtually all living cells, that can degrade RNA molecules through both endonucleolytic and exonucleolytic activity. They are remarkably stable enzymes and their control is of paramount importance. It is incumbent upon the investigator to ensure that both equipment and reagents are purged of nucleases from the onset of the experiment. The most straightforward procedure to this end is for the lab to develop a detailed standard operating procedure (SOP), including a checklist, which each person in the lab is obligated to follow. Protocols for the isolation of RNA begin with cellular lysis mediated by buffers that typically fall into one of two categories depending on the required degree of cellular disruption and concomitant RNase inhibition: (1) those consisting of harsh chaotropic agents such as one of the guanidinium salts, which disrupt the plasma membrane and subcellular organelles and which simultaneously inactivate RNase; and (2) those that gently solubilize the plasma membrane while maintaining nuclear and other organelle integrity, such as the nonionic, hypotonic lysis buffers. The disadvantage of using gentle lysis buffers is that the lysis buffer alone is not sufficiently chaotropic to fully inhibit RNase activity; and the drawback of harsh chaotropic procedures is that the investigator is not able to discriminate between RNAs from various subcellular compartments once various subpopulations have been mixed. There are many commercial products available for RNase control. Failure to do so will inevitably result in a useless sample of degraded RNA and marked reduction in lab productivity.

Background and objective Andaliman fruit (Zanthoxylum acanthopodium) is a well-known spice antioxidant in Northern Sumatera (Indonesia). The cellular activity requires antioxidants in counteracting free radicals. The cellular proteins that play a role in development, proliferation, differentiation and embryonic processes in the human placenta are NOTCH1 and Hes1. The aim of this research was to analyze the expression of NOTCH1 and Hes1 genes after administering nano herbal andaliman to the trophoblast cells of the human placenta. Materials and methods HTR8 trophoblast cells were divided into two groups, namely, control and treatment (nano herbal andaliman). RNA isolation, reverse transcription and RT-PCR (real-time polymerase chain reaction) were performed to analyze the NOTCH1 and Notch target gene (Hes1) expressions. The NOTCH1 and Hes1 gene expressions were quantified using the CT method (2-ΔΔCT) and normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expressions. Results Nanoherbal andaliman reduced the expression of NOTCH1 genes in the human placental trophoblast. However, it increased the expression of Hes1 when the incubation time was 16 hrs. Conclusion Nanoherbal andaliman decreases the expression of genes that are crucial in hypoxia and free radicals in the placenta, namely, NOTCH1 and Hes1 increased after incubation for 16 hrs. Therefore, this herb needs to be evaluated further.

Preeclampsia is a complication of pregnancy characterized by high blood pressure and organ damage after 20 gestational weeks. It is associated with high maternal and fetal morbidity and mortality. However, at present, there is no effective prevention or treatment for this condition. Previous studies have revealed that plasma exosomal mirnas from pregnant women with preeclampsia could serve as biomarkers of pathogenic factors. However, the roles of plasma exosomal mirnas in preeclampsia with severe features (sPE), which is associated with poorer pregnancy outcomes, remain unknown. Thus, the aims of this study were to characterize plasma exosomal miRNAs in sPE and explore the related pathogenic mechanisms using bioinformatic analysis. Plasma exosomes were isolated using a mirVana RNA isolation kit. the exosomal miRNAs were detected using high-throughput sequencing and the mirnas related to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms were analyzed using the clusterprofiler package of R. Fifteen miRNAs exhibited increased expression and fourteen miRNAs exhibited reduced expression in plasma exosomes from women with sPE as compared to normal pregnant women. Further, gene set enrichment analysis revealed that the differentially expressed plasma exosomal miRNAs were related to the stress response and cell junction regulation, among others. In summary, this study is the first to identify the differentially expressed plasma exosomal miRNAs in sPE. These findings highlight promising pathogenesis mechanisms underlying preeclampsia.

Since the start of the coronavirus disease 2019 (COVID-19) pandemic, molecular diagnostic testing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has faced substantial supply chain shortages and noteworthy delays in result reporting after sample collection. Supply chain shortages have been most evident in reagents for RNA extraction and rapid diagnostic testing. This study explored the kinetic limitations of extraction-free rapid cycle quantitative real-time RT-PCR for SARS-CoV-2 virus detection using the commercially available capillary-based LightCycler. After optimizing for time and reaction conditions, a protocol for sensitive and specific quantitative RT-PCR of SARS-CoV-2 RNA from nasopharyngeal swabs in

Purity and integrity are two important criteria for any RNA extraction process to qualify the RNA for meaningful gene expression analysis. This study compares four commercially available RNA extraction kits using silica membrane and magnetic bead separation methods. The performance was evaluated in terms of both quantity (total RNA amount in μg/μl) and purity (260/280 ratio). The concentration and purity of each kit was significantly different from those of the others (p

Background Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. Results We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. Conclusions We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.

Objective: Peri-implantitis is a condition resulting in destructive inflammation in the peri-implant soft tissue barrier. Clinically, it demonstrates vast clinical differences to periodontitis that suggest distinct inflammatory mechanisms. Implant-derived titanium particles (i-TiPs) frequently found around diseased implants appear to alter the microenvironment and confer resistance to antibiotic treatments. Studies in orthopedic implants have demonstrated potent inflammatory responses to i-TiPs involving a variety of cell types in aseptic conditions. Nonetheless, the genetic programs of cells surveilling and supporting the peri-implant soft tissue barrier in response to the combined challenges of biomaterial degradation products and oral bacteria are poorly defined. Thus, we studied gene expression specific to oral peri-implant inflammatory disease. Methods: Peri-implant tissues were collected from healthy or diseased implants (N = 10) according to the 2018 classification criteria. Following RNA extraction and purification, a gene-level view of the transcriptome was obtained via a next-generation transcriptome-wide microarray profiling workflow (Clariom S; Applied Biosystems) that covers >20,000 well-annotated genes. A discovery analysis assessed global differential expression of genes and identified pathways in peri-implant health versus disease. Results: Genes involved in the endosomal-lysosomal pathway, such as actin polymerization, were strongly upregulated in diseased tissues (P < .05), proposing increased intracellular activities in response to bacteria and i-TiPs. Cellular respiration pathways involved in oxidative stress were highly transcribed in all peri-implant samples, suggesting that implant-specific factors may trigger a constant state of oxidative stress. Conclusion: Within the limitations of this discovery study, expressive upregulation of genes in the endosomal-lysosomal and oxidative stress pathway suggests that inflammation related to receptor-driven responses to extracellular signals, such as i-TiPs and pathogens, may have a crucial role in peri-implantitis. Results warrant external replication in validation cohorts. Knowledge Transfer Statement: Our findings regarding physiologic processes affected by peri-implantitis could advance knowledge of the mechanisms and consequences of the disease. Understanding the cellular programs that partake in peri-implant inflammation has the potential to translate to novel treatment strategies for patients with peri-implantitis.

Background: Oral Squamous Cell Carcinoma (OSCC) is the most common malignancies which accounts for 90% of oral cancer worldwide. The 5 year survival rate of OSCC is not more than 60% due to tumor metastasis and subsequent recurrence. Aim: To compare the gene expression of ITG β-1, MMP9 and Vimentin in OSCC tissue samples and normal tissue samples and to correlate the expression levels of these molecules with the pathological grading and survival in OSCC patients. This would facilitate the understanding of EMT in OSCC progression thereby targeting this pathway for treatment of OSCC patients in near future. Materials and Methods: 10 OSCC samples as well as normal healthy samples were collected and RNA isolation was done using TRIR kit, and then subjected to cDNA synthesis using ITG β-1, MMP9 and Vimentin primers. Real time PCR was performed using gene specific primers at 40 cycles. The results were retrieved, tabulated and analyzed. Results: The current research results revealed that there were up regulation of mRNA expression in ITG β-1, MMP9 and Vimentin in OSCC patients than in healthy individuals. On comparison, MMP9 showed highest mRNA expression levels than ITG β-1 and Vimentin Conclusion: Over expression of ITG B-1, MMP9, Vimentin plays a crucial role in progression of oral cancer and targeting EMT molecules could be an effective targeted approach for OSCC.

RNA isolation from bacteria is technically difficult due to the RNA characteristic of labile and vulnerable degradation. Many reagents were explored for cellular lysis and complete inhibition of RNase. However, the available methods for RNA isolation are either of low efficiency or time-consuming. Here, we developed a rapid and accessible protocol for RNA isolation that combined a simplified cell lysis and RNA release by formamide-based solution and RNA purification by chitosan-modified silica membrane for the first time. With this method, we obtained about ~ 28 μg of total RNA from 108 Escherichia coli cells. The entire procedure can be done within 15 min without redundant pipetting steps. The purity of extracted RNA was comparable to that of commercial kits, but the cost was much lower. Furthermore, the yielded RNA was successfully used in downstream enzymatic reactions, such as reverse transcription and quantitative real-time PCR. This new method would be of benefit for an extensive range of gene expression analyses in bacterial organisms.

This study aimed to evaluate the effect of micro-osteoperforations (MOPs) on the gene expression profile of the periodontal ligament (PDL) of orthodontically moved teeth. Fifteen participants were randomly assigned into two groups: tooth movement only (Tr1, n = 7) and tooth movement supplemented with MOPs (Tr2, n = 8). In each subject, orthodontic tooth movement (OTM) was performed on premolar in one side, while no force was applied on contralateral premolar (Unt, n = 15). Seven days after loading, premolars were extracted for orthodontic reasons. RNA extraction from PDL and subsequent RNA-sequencing were performed. False discovery rates (Padj

The worldwide coronavirus disease 2019 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce 2 pH-sensitive “LAMPshade” dyes as novel readouts in an isothermal Reverse Transcriptase Loop-mediated isothermal AMPlification amplification assay for severe acute respiratory syndrome coronavirus 2 RNA. The resulting JaneliaLAMP assay is robust, simple, inexpensive, and has low technical requirements, and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.

Background One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol. Methods We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R2 = 0.89). Conclusions These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable.

Background SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. However, robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples. Results Stool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Viral particles were concentrated by ultrafiltration, RNA was extracted, and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes. The RNA extraction procedure we used allowed for the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool. Conclusions Here we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as FMT.

Assessing male reproductive toxicity of environmental and therapeutic agents relies on the histopathology of the testis and epididymis in a pre-clinical setting. Animal histopathology poorly correlates with human sperm parameters, and none of these current methods are strong indicators of sperm health or reproductive potential. Therefore, there is an urgent need to identify a translatable, non-invasive and reliable approach to monitor environmental and therapeutic agents' effects on male reproductive health. mRNA sequences were analyzed in mouse, rat and human sperm samples to identify sperm transcriptomic similarities across species that could be used as biomarkers to predict male reproductive toxicity in animal models. Semen specimens were collected from men aged 18 to 55 years with proven fertility. Rat and mouse semen specimens were collected via needle punctures of the cauda epididymides. Sperm RNAs were extracted using an optimized sperm RNA isolation protocol and subjected to polyA-purified mRNA-sequencing. Bioinformatics analyses, including differential abundance and gene set enrichment analysis, were used to investigate the biological and molecular functions of all shared and differentially abundant transcripts across species. Transcriptome profiling identified 6,684 similarly expressed transcripts within the three species of which 1,579 transcripts were found to be involved in spermatogenic functions. Our findings have shown that sperm transcriptome is highly species dependent, however, there are some key similarities among transcripts that are required for fertility. Based on these similarities, sperm mRNA biomarker may be developed to monitor male reproductive toxicity where rodent models would make suitable laboratory substitutes for human.

Transcriptomic information provides fundamental insights into biological processes and can be used to determine gene expression in cell, tissue, or organism under specific physiological conditions, or in response to any environmental perturbation. Extraction of high quality RNA is a challenging step mainly in non-traditional organisms, and protocols for preservation and isolation need to be adjusted in many cases. In the present work, we aimed to develop a protocol for preservation and isolation of high-quality and quantity of RNA from blood and liver tissues of Caiman latirostris. Three preservation methods were tested: 1) flash freezing (LN2) and storage at –80°C; 2) RNAlater® conservation with progressive cooling up to –80°C); 3) preservation in TRIzol® reagent, flash freezing in LN2 and storage at –80°C. Methods 1 and 2 were tested for liver, while 2 and 3 for blood. Our results showed that both preservation methods resulted in excellent outcomes for liver samples. For blood samples however, TRIzol® preservation was an efficient procedure for adequate RNA quality, quantity, and integrity, while conservation in RNAlater® solution was inadequate in both quality and quantity for an optimal RNA extraction. Appropriate protocols were established for each tissue and are being used now for transcriptomic studies in this sentinel organism.

Autophagy process is an important defense mechanism against intracellular infection. This process plays a critical role in limiting the development of Toxoplasma gondii. This study aimed to investigate the effects of T. gondii profilin and tachyzoites on the expression of autophagy genes. PMA-activated THP-1 cell line was incubated with T. gondii profilin and tachyzoites for 6 h. After RNA extraction and cDNA synthesis, the expression of Atg5, Atg7, Atg12, and LC3b was evaluated using real-time PCR. The results revealed statistically significant downregulation of Atg5 for 1.43 (P-value = 0.0062) and 4.15 (P-value = 0.0178) folds after treatment with T. gondii profilin and tachyzoites, respectively. Similar to Atg 5, Atg 12 revealed a statistically significant downregulation for profilin (1.41 fold; P-value = 0.0047) and T. gondii tachyzoites (3.25 fold; P-value = 0.011). The expression of Atg7 elevated in both T. gondii profilin (2.083 fold; P-value = 0.0087) and tachyzoites (1.64 fold; P-value = 0.206). T. gondii profilin and tachyzoites downregulated (1.04 fold; P-value = 0.0028) and upregulated (twofold; P-value = 0.091) the expression of LC3b, respectively. Our findings suggest that T. gondii and profilin may manipulate autophagy via preventing from the formation of Atg5-12-16L complex to facilitate replication of T. gondii and development of toxoplasmosis.

The phototrophic bacterium Rhodopseudomonas palustris is emerging as a promising biotechnological chassis organism, due to its resilience to a range of harsh conditions, a wide metabolic repertoire, and the ability to quickly regenerate ATP using light. However, realization of this promise is impeded by a lack of efficient, rapid methods for genetic modification. Here, we present optimized tools for generating chromosomal insertions and deletions employing electroporation as a means of transformation. Generation of markerless strains can be completed in 12 days, approximately half the time for previous conjugation-based methods. This system was used for overexpression of alternative nitrogenase isozymes with the aim of improving biohydrogen productivity. Insertion of the pucBa promoter upstream of vnf and anf nitrogenase operons drove robust overexpression up to 4000-fold higher than wild-type. Transcript quantification was facilitated by an optimized high-quality RNA extraction protocol employing lysis using detergent and heat. Overexpression resulted in increased nitrogenase protein levels, extending to superior hydrogen productivity in bioreactor studies under nongrowing conditions, where promoter-modified strains better utilized the favorable energy state created by reduced competition from cell division. Robust heterologous expression driven by the pucBa promoter is thus attractive for energy-intensive biosyntheses suited to the capabilities of R. palustris. Development of this genetic modification toolset will accelerate the advancement of R. palustris as a biotechnological chassis organism, and insights into the effects of nitrogenase overexpression will guide future efforts in engineering strains for improved hydrogen production.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater has been reported as a result of fecal shedding of infected individuals. In this study, the occurrence of SARS-CoV-2 RNA was explored in primary-treated wastewater from two municipal wastewater treatment plants in Quintana Roo, Mexico, along with groundwater from sinkholes, a household well, and submarine groundwater discharges. Physicochemical variables were obtained in situ, and coliphage densities were determined. Three virus concentration methods based on adsorption-elution and sequential filtration were used followed by RNA isolation. Quantification of SARS-CoV-2 was done by RT-qPCR using the CDC 2020 assay, 2019-nCoV_N1 and 2019-nCoV_N2. The Pepper mild mottle virus, one of the most abundant RNA viruses in wastewater was quantified by RT-qPCR and compared to SARS-CoV-2 concentrations. The use of three combined virus concentration methods together with two qPCR assays allowed the detection of SARS-CoV-2 RNA in 58% of the wastewater samples analyzed, whereas none of the groundwater samples were positive for SARS-CoV-2 RNA. Concentrations of SARS-CoV-2 in wastewater were from 1.8 × 103 to 7.5 × 103 genome copies per liter (GC l-1), using the N1 RT-qPCR assay, and from 2.4 × 102 to 5.9 × 103 GC l-1 using the N2 RT-qPCR assay. Based on PMMoV prevalence detected in all wastewater and groundwater samples tested, the three viral concentration methods used could be successfully applied for SARS-CoV-2 RNA detection in further studies. This study represents the first detection of SARS-CoV-2 RNA in wastewater in southeast Mexico and provides a baseline for developing a wastewater-based epidemiology approach in the area.

Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. We recently developed a full-length nascent RNA sequencing method in the model plant Arabidopsis that simultaneously detects RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale. This method allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally. Here we describe a detailed protocol for this method called FLEP-seq (full-length elongating and polyadenylated RNA sequencing) that is applicable to plants. Library production requires as little as one nanogram of nascent RNA (after rRNA/tRNA removal), and either Nanopore or PacBio platforms can be used for sequencing. We also provide a complete bioinformatic pipeline from raw data processing to downstream analysis. The minimum time required for FLEP-seq, including RNA extraction and library preparation, is 36 h. The subsequent long-read sequencing and initial data analysis ranges between 31 and 40 h, depending on the sequencing platform.

Influenza A virus (IAV) was a neglected swine pathogen in South America before the 2009 H1N1 pandemic (A(H1N1)pdm2009). The A(H1N1)pdm2009 strain has widely spread among the Chilean swine population and co-circulates with endemic H1N2 and H3N2 viruses. The presence of IAV as a swine pathogen in Chilean swine before the 2009 pandemic is unknown. To understand the IAV in swine prior to 2009, a retrospective study of samples from pigs affected with respiratory diseases was conducted. Ninety formalin-fixed and paraffin-embedded (FFPE) lung tissues belonging to 21 intensive pig production companies located in five different administrative regions of Chile, collected between 2005 and 2008, were evaluated. The tissues were tested by immunohistochemistry (IHC), identifying that 9 out of 21 farms (42.8%) and 31 out of 90 (34.4%) samples were IAV positive. Only three out of the 31 IHC positive samples were positive upon RNA extraction and rtRT-PCR analysis. Partial nucleotide sequences were obtained from one sample and characterized as an H3N2 subtype closely related to a human seasonal H3N2 IAVs that circulated globally in the mid-90s. These results indicate that IAV was circulating in swine before 2009 and highlight the value of conducting retrospective studies through genomic strategies to analyze historical samples. This article is protected by copyright. All rights reserved.

In captivity, cardiovascular diseases are common in grey parrots. The diagnosis of these diseases in living birds is difficult, and new diagnostic possibilities would be desirable. The heart is an important endocrine organ in which cardiomyocytes synthetise B-type natriuretic peptide (BNP) and release it into the bloodstream. This hormone has a significant role in cardiovascular and body fluid regulation. The blood concentration of BNP is used in human medicine and small animal medicine as a diagnostic tool in the identification of heart diseases and as a prognostic marker for the risk of mortality. The nucleotide and amino acid sequence of BNP was described in Congo (n = 4) and Timneh (n = 3) grey parrots by PCR after RNA isolation from the atria and ventricles. The results showed a high similarity between the nucleotide sequences of the grey parrots’ BNP and the already known sequence of this hormone in chickens. The amino acid sequence of the mature peptide region is consistent in these three species. BNP plasma concentration could be a possible blood parameter for identifying clinically manifest cardiovascular diseases in grey parrots as it is in other species.

The emergence of a novel corona virus was seen in the human population since December 2019. The Covid-19 is posing a major burden on society. Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has since spread across the globe. Thus, early and correct diagnosis is very crucial to reduce its spread. So, most sensitive and specific method available to diagnose the Covid-19 is real-time reverse transcriptase PCR (qRT-PCR). A better qRT-PCR result depends on the quality of the extracted RNA. Recently, many commercial kits have become available for extraction of RNA from specimens. The goal of this study was to compare robustness or clinical performance of manual kits available to us (i) MDI Viral RNA Extraction Mini-prep Kit and 2 automated extraction systems such as (i) MagNA Pure 24 Roche (ii) Nextactor NX-48 Genolution. We used 48 pre-examined nasopharyngeal and oropharyngeal swab samples with low, medium and high cycle threshold (Ct) values for this study and extraction was done in pooling of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10. Through this work, we conclude that MagNA Pure 24 Roche and MDI Viral RNA Extraction Mini-prep kit showed better result as compared to Nextactor NX-48 Genolution. Keywords: SARS-CoV-2, qRT-PCR, Extraction, Diagnosis.

Isolation of an adequate quantity of high-quality RNA is a crucial step in gene expression analysis. Difficulties in sampling and RNA extraction of field-grown crops, especially from roots, are substantial obstacles to understanding actual gene expression in the field. We examined the effectiveness of the RNA extraction method (FRP) using pot- and field-grown soybean roots. High quantity and quality of total RNA could extract by the FRP method from both pot- and field-grown soybean roots at different growth stages. To determine the influence of root washing during the sampling process, roots were washed with water for 5 s, 1 min, and 5 min, then analyzed for aquaporin gene expression levels. Root washing for 5 min affected gene expression of some aquaporins. To obtain RNA that reflects the correct gene expression profile, it is necessary to minimize the effects of the sampling process.

Introduction. When conducting PCR (polymerase chain reaction) testing of biospecimens for SARS-CoV-2 RNA at the beginning of the COVID-19 pandemic, the laboratory service in Russia and foreign countries encountered problems related to the accuracy of diagnostics and obtaining false negative, false positive, and dubious results. The objective of this work was to analyze current literature on the problem of false positive and dubious results of RT-PCR testing for COVID-19. Material and methods. We selected Russian and foreign English-language publications devoted to organization of laboratory diagnostics of the novel coronavirus disease, challenges of PCR testing for SARS and MERS, and general issues of DNA contamination in a PCR laboratory for 2012–2020. We also reviewed current regulations and guidelines for COVID-19 diagnostic testing. Results. The analysis of factors leading to contamination of specimens with nucleic acids in the laboratories performing massive COVID-19 PCR testing during the pandemic showed that the main reasons for contamination included a large number of tests, accumulation of samples in the laboratory, and the increased amount of wastes containing amplification products. Cross-contamination occurs due to technical errors in the course of laboratory manipulations at the stages of sample preparation and inactivation, RNA isolation, and addition of cDNA/RNA or positive control samples to the reaction mixture. Pollution of laboratory working areas with amplicons arising from the opening of tubes and plates containing PCR products is the main cause of total contamination in the laboratory. Signs of cross-contamination include the increase in the proportion of positive samples with low threshold cycle values and detection of a positive signal from negative control samples at RNA isolation and amplification stages. A positive result for all samples in a round, including negative control samples, is a marker of “total contamination” in the laboratory. In addition to contamination, formation of nonspecific PCR products at late reaction cycles and nonspecific fluorescence of the reaction mixture, which occurs when reagent storage temperatures are not observed, may also lead to false positive results. Conclusion. To prevent contamination in a PCR laboratory, strict control over the flow of test samples and medical wastes, regular analysis of the frequency of positive test results, and mandatory laboratory quality control of testing and DNA/RNA contamination are compulsory.

Acquired resistance to glucocorticoids (GCs) is an obstacle to the effective treatment of leukemia, but the molecular mechanisms of steroid insensitivity have not been fully elucidated. In this study, we established an acquired GC-resistant leukemia cell model and found a long noncoding RNA, HOTAIRM1, was overexpressed in the resistant cells by transcriptional profiling, and was higher expressed in patients with poor prognosis. The whole-genome-binding sites of HOTAIRM1 were determined by ChIRP-seq (chromatin isolation by RNA purification combined with sequencing) analysis. Further study determined that HOTAIRM1 bound to the transcriptional inhibitory region of ARHGAP18 and repressed the expression of ARHGAP18, which led to the increase of RHOA/ROCK1 signaling pathway and promoted GC resistance through antiapoptosis of leukemia cells. The inhibition of ROCK1 in GC-resistant cells could restore GCs responsiveness. In addition, HOTAIRM1 could also act as a protein sequester to prevent transcription factor AML1(acute myeloid leukemia 1) from binding to the regulatory region of ARHGAP18 by interacting with AML1. At last, we also proved AML1 could directly activate the expression of HOTAIRM1 through binding to the promoter of HOTAIRM1, which enriched the knowledge on the regulation of lncRNAs. This study revealed epigenetic causes of glucocorticoid resistance from the perspective of lncRNA, and laid a foundation for the optimization of glucocorticoid-based leukemia treatment strategy in clinic.

Purpose.Grapevines (Vitisspp.) are affected by many viral diseases which cause serious pathological problems. GLRaV-3 is among the most widespread leafroll viruses, while Grapevine Fanleaf Virus (GFLV) is a destructive pathogen which reduces the lifespan of grapevine. Considering the impact and the spread of these diseases, our objective was to analyse the presence of these two viruses in several grapevine varieties in grapevine collection at ATTC Vlore. Data gathered from plant pathogens serve to better understand and prevent the spread of pathogens, as a mandatory rule for the quality control of certified plant material during vegetative propagation.Method.The presence of two common viruses were tested using virus specific primers; LC1/LC2 primer pair designed from the hHSP70 gene for detecting Grapevine Leafroll-associated Virus-3 (GLRaV3) and C3390/H2999 primer pair, designed from coat protein coding regions for detecting Grapevine Fanleaf Virus (GFLV), in six varieties; ‘Merlot’, ‘Kallmet’, ‘Shesh i zi’, ‘Shesh i bardhё’, ‘Debinё’, and ‘Pulёz’, provided through a randomised sampling procedure. One Step Reverse Transcription Polymerase Chain Reaction assay was used to detect the viral presence.Resultsshowed a high (100%) prevalence of GLRaV3 virus in all of analysed samples, as the most frequent among the two pathogens. Analysis for of GFLV virus showed low infection rate, being present in only one sample.Conclusions.We herein show an efficient, fast and reproducible method for detecting grapevine viruses through one step RT-PCR. Our results suggest that sampling of the infected plant material should be avoided due to the presence of viral infections.

More than 90% of the human genome is transcribed into noncoding RNAs, but their functional characterization has lagged behind. A major bottleneck in the understanding of their functions and mechanisms has been a dearth of systematic methods for identifying interacting protein partners. There now exist several methods, including identification of direct RNA interacting proteins (iDRiP), chromatin isolation by RNA purification (ChIRP), and RNA antisense purification, each previously applied towards identifying a proteome for the prototype noncoding RNA, Xist. iDRiP has recently been modified to successfully identify proteomes for two additional noncoding RNAs of interest, TERRA and U1 RNA. Here we describe the modified protocol in detail, highlighting technical differences that facilitate capture of various noncoding RNAs. The protocol can be applied to short and long RNAs in both cultured cells and tissues, and requires ~1 week from start to finish. Here we also perform a comparative analysis between iDRiP and ChIRP. We obtain partially overlapping profiles, but find that iDRiP yields a greater number of specific proteins and fewer mitochondrial contaminants. With an increasing number of essential long noncoding RNAs being described, robust RNA-centric protein capture methods are critical for the probing of noncoding RNA function and mechanism. This protocol describes how to identify direct protein targets of noncoding RNAs—Xist, TERRA and U1—and outlines modifications specific to each noncoding RNA and its partners.

Background: Research on stem cells has become one of the most promising and advanced scientific topics. Nevertheless, there is a lack of this type of research in the field of forensic diagnostics. Age estimation is one of the main worked-on issues in forensic practice and research. Still, there is a need to explore new complementary reliable methods. This study aimed to detect the possibility of age estimation via age-associated alteration of RIF-1 (Replication timing regulatory factor 1), TCL1A (T-cell leukemia/lymphoma-1A), and TERT (Telomerase reverse transcriptase) gene expression in rats bone marrow stem cells. Methods: Isolation of bone marrow mesenchymal stem cells from rats were undertaken at different ages; 2, 4, 6, 8, 10, and 12 weeks old. RNA extraction was performed then total RNA was reverse transcribed to cDNA to detect the quantitative expression of RIF-1, TLC1A, and TERT by using the qRT-PCR then immunoblotting. Results: There were statistically significant increases in RIF1 and TCL1A mRNA and protein expression and a decrease in TERT mRNA and protein expression with age. Conclusion: Our results showed that the mRNA and protein expression of RIF-1, TCL1A, and TERT in bone marrow cells of rats could be useful indicators for age estimation in forensic practice.

Background Noncoding RNAs such as circular RNAs (circRNAs) are abundant in the human body and influence the occurrence and development of various diseases. However, the biological functions of circRNAs in colorectal cancer (CRC) are largely unknown. Methods RT-qPCR was used to detect the expression of circRNAs and mRNA in CRC cells and tissues. Fluorescence in situ hybridization (FISH) was used to analyze the location of circSPARC. Function-based experiments were performed using circSPARC knockdown and overexpression cell lines in vitro and in vivo, including CCK8, colony formation, transwell and metastasis models. Mechanistically, luciferase reporter assay, western blots, RNA immunoprecipitation (RIP), Chromatin isolation by RNA purification (ChIRP) and immunohistochemical stainings were performed. Results CircSPARC was upregulated in both the tissues and plasma of CRC patients. High expression of circSPARC was associated with advanced TNM stage, lymph node metastases, and poor survival. Silencing circSPARC inhibited CRC cell migration and proliferation in vitro and vivo. Mechanistically, circSPARC sponged miR-485-3p to upregulate JAK2 expression and ultimately contribute to the accumulation of phosphorylated (p)-STAT3. Besides, circSPARC recruited FUS, which facilitated the nuclear translocation of p-STAT3. Conclusions These findings suggest that circSPARC might serve as a potential diagnostic and prognostic biomarker and a therapeutic target for CRC treatment by regulating JAK2/STAT3 pathway.

MicroRNA can be found in various body fluids, including breast milk. MicroRNA may be transferred from mother to infant via breast milk and potentially regulate the development of the infants immune system on a post-transcriptional level. This study aimed to determine the microRNA extraction efficiency of five RNA extraction kits from human skim milk samples. Their efficiency was determined by comparing microRNA concentrations, total RNA yield and purity. Furthermore, hsa-miR-148a-3p expression and the recovery of an exogenous control, cel-miR-39-3p, were quantified using qPCR. Each kit extracted different amounts of microRNA and total RNA, with one kit tending to isolate the highest amount of both RNA species. Based on these results, the extraction kit ReliaPrep (TM) miRNA Cell and Tissue Miniprep System from Promega was found to be the most appropriate kit for microRNA extraction from human skim milk. Moreover, further research is needed to establish a standardized protocol for microRNA extraction from breast milk. Funding Agencies|Lisa och Johan Gronbergs Stiftelse

AimCruciate ligaments (CLs) of the knee joint are commonly injured following trauma or ageing. MicroRNAs (miRs) are potential therapeutic targets in musculoskeletal disorders. This study aimed to 1) identify if wild-stock house (WSH) mice are an appropriate model to study age-related changes of the knee joint and 2) investigate expression of miRs in ageing murine CLs.MethodsKnee joints were collected from 6 and 24 months old C57BL/6 and WSH mice (Mus musculus domesticus) for histological analysis. RNA extraction and qPCR gene expression were performed on CLs in 6, 12, 24, and 30 month WSH old mice. Expression of miR targets in CLs was determined, followed by analysis of predicted mRNA target genes and Ingenuity Pathway Analysis.ResultsHigher CL and knee OARSI histological scores were found in 24 month old WSH mice compared to 6 and 12 month old C57BL/6 and 6 month old WSH mice (p< 0.05). miR-29a and miR-34a were upregulated in 30 month-old WSH mice in comparison to 6, 12 and 24-month-old WSH mice (pConclusionThe findings of this study support WSH house mice as an accelerated ageing model of the murine knee joint. This study also indicated that miR-29a and 34a may be important regulators of COL1A1 gene expression in murine CLs.

BackgroundSensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines.MethodsHere, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here.ResultsMost RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105copies of viral genome/μl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost.ConclusionsWith extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct

Understanding the essential processes of heredity, mechanisms of metabolism, and evolutionary traits of this ginkgo (Ginkgo biloba L.) in terms of molecular biology is necessary. The extraction of high-quality and high-integrity RNA from ginkgo is one of the basic techniques of molecular biology. Therefore, this study aimed to compare the efficacy of the six commonly used RNA extraction methods for detection. TRIzol, modified TRIzol, cetyltrimethylammonium bromide (CTAB), modified CTAB, sodium dodecyl sulfate (SDS), and RNeasy Plant Mini Kit (Qiagen) methods, were evaluated for their capacity to extract high-quality and high-quantity RNA from the embryo and endosperm of ginkgo. The advantages and disadvantages of each method were analyzed to evaluate the isolation process. The optimized modified CTAB method for embryo prevented the precipitation of RNA for a long period and reduced complex separation and precipitation processes. In addition, the modified TRIzol method is more effective in endosperm. Obvious differences in RNA quality and quantity were observed between the embryo and endosperm when the same method is used. Modified TRIzol, modified CTAB, SDS and RNeasy Plant Mini Kit (Qiagen) methods were applied to RNA extraction from other different ginkgo tissues. The modified CTAB is efficient and recommended for the isolation of good-quality total RNA from all tissues with reduced chemical use, costs, and RNA loss. The results of quantitative real-time PCR confirmed that modified CTAB methods could obtain showing low Ct values and high amplification efficiency and enabled the successful isolation of RNA from all tissues.

Background The Eurofins GeneScan Technologies’ VIRSeek SARS-CoV-2 Mplex kit is a RT (reverse transcription) real-time polymerase chain reaction (RT-qPCR) assay for the detection of two targets on the N-gene (nucleocapsid) of SARS-CoV-2. An extraction control, that allows monitoring of the extraction procedure and PCR inhibition, is included. Objective In silico analysis and wet testing showed inclusivity and exclusivity of the assay. The complete workflow starting from surface swabbing (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS-CoV-2 Mplex), and evaluation with FastFinder was validated in comparison to the CDC method for detection of SARS-CoV-2 on stainless steel. Method In silico analysis was performed by using the MFOLD online program. The matrix study was performed for stainless steel inoculated with SARS-CoV-2 isolated from the first documented US case of a traveler from Wuhan, China. Results For inclusivity, 15 764 sequences were analyzed and all mismatches (0.37% of the sequences had single mismatches) were considered non-critical. Cross reactivity for closely related viruses and background organisms was performed, resulting in correct exclusion of all. No significant differences were observed for the probability of detection (POD) study when comparing to the CDC method. Conclusions Results of the inclusivity and exclusivity study show that the assay is specific for detection of SARS-CoV-2. The POD study showed no statistically significant difference compared to the CDC reference method, results were identical for the uninoculated and the high level. For the fractional recovery level, the candidate method detected 9/17 samples leading to a POD of 0.47, the reference method detected 11/20 samples leading to a POD of 0.55. Highlight The complete workflow starting from swabbing of the surface (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS CoV-2 Mplex) and evaluation with FastFinder was validated in comparison to the US Centers for Disease Control and Prevention method for detection of SARS-CoV-2 on Stainless Steel.

Обоснование. Альтернативный путь дыхания митохондрий растений не связан с синтезом АТФ и, следовательно, не контролируется непосредственно энергетическим статусом клетки. Альтернативный дыхательный путь включает в себя ротенон нечувствительные NAD(Р)Н дегидрогеназы II типа, расположенные как на внешней, так и на внутренней поверхности внутренней мембраны митохондрий, убихинон и альтернативную оксидазу (AOX). Предполагается, что альтернативные NAD(P)H дегидрогеназы имеют функции, сходные с функциями AOX, среди которых термогенез, предотвращение образования АФК, окисление избытка восстановителей для продолжения метаболических путей и др. Публикаций об успешном подавлении экспрессии NDB2 пока недостаточно. Например, показано, что растения арабидопсиса, лишенные и AOX1a и NDB2 были более чувствительны к комбинированной засухе и повышенному освещению, в то время как растения, гиперэкспрессирующие эти гены, демонстрировали повышенную устойчивость и способность к постстрессовому восстановлению. Цель. Целью данной работы являлось изучить, как подавление экспрессии NDB2 в гетеротрофных клетках суспензионной культуры арабидопсиса повлияет на экспрессию других генов альтернативного пути дыхания, разобщающих белков, а также генов белков теплового шока в не стрессовых условиях. Материалы и методы. Для выделения РНК отбирали по 5 мл клеток суспензионных культур. РНК выделяли с помощью реактивов из набора GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, Литва), согласно инструкции производителя. Синтез первой цепи кДНК осуществляли с использованием набора реактивов Thermo Scientific (Литва), согласно рекомендациям фирмы производителя. ПЦР-РВ проводили на приборе CFX96™ Real-Time PCR Detection System (Bio-Rad, США), используя набор реактивов qPCR mix-HS SYBR (Евроген, Россия), согласно инструкции производителя. Анализ данных ПЦР-РВ проводили с помощью программного обеспечения SFX Manager (Bio-Rad, Германия). Все эксперименты проводились в двух аналитических и трех биологических повторностях. В качестве референсного гена использовали ген, кодирующий глицеральдегид-3-фосфат дегидрогеназу – GAPD. Результаты. Количество мРНК гена NDB2 в клетках суспензионной культуры линии AS5 было снижено в 8,2 раза по сравнению с контролем Col-0. Подавление экспрессии гена NDB2 в клетках суспензионной культуры арабидопсиса линии AS5 приводило к увеличению количества транскриптов не только гена NDB4, но и NDB1, а также NDA2 и NDC1; в то же время экспрессия генов NDB3 и NDA1 снижалась по сравнению с клетками Col-0. Мы не обнаружили изменения уровней экспрессии генов AOX1a, AOX1b и AOX1d в клетках линии AS5 по сравнению с контролем, однако количество транскриптов гена AOX1c несколько увеличивалось. При анализе уровней экспрессии генов, кодирующих разобщающие белки, было обнаружено увеличение экспрессии гена UCP1 в клетках линии AS5. В клетках суспензионной культуры линии AS5 повышались уровни экспрессии всех исследуемых нами генов БТШ, кроме HSP17.7. Заключение. Таким образом, полученные результаты предполагают, что подавление экспрессии гена NDB2 в гетеротрофных клетках арабидопсиса в отсутствие стресса изменяет редокс-статус клетки, что, в свою очередь, приводит к изменениям уровней накопления транскриптов других генов NAD(Р)Н дегидрогеназ и увеличению экспрессии генов БТШ, при этом экспрессия ключевых генов AOX не изменяется.

Nasopharyngeal myasis in horses with Rhinoestrus species has been associated with irritation of nasal cavities; pharynx and olfactory nerve injuries have varying degrees of clinical signs of symptoms until the death of animals. The aim of this study is to determine the cellular immune response against the infestation of Rhinoestrus spp. Therefore, the collection of Rhinoestrus spp. and its turbinates’ under aseptically condition. Morphological and molecular identification of the larvae to determine the larval species. Then; the turbinates’ subjected to RNA extraction against Tumor necrosis factor- alpha (TNF- α) through using of quantitative Real- time PCR. In the first larval instar (L1); the intensity of the larvae was recorded in relation with the genes expression as the animals had 1–10 larvae the TNF-α means was 5.55 ± 0.25. The animals that harbored 11–20 L1 the genes were 10.85 ± 0.14 in TNF- α. In the second and third larval instar (L2); the intensity of the larvae was recorded in the relation with the gene expression. In conclusion, the gene was expressed regularly against the infestation to Rhinoestrus usbekistanicus that provoked with the production of several immunological cells such as mast cell; eosinophils; macrophages and lymphocytes.

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods’ sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled “Sewage, Salt, Silica and SARS-CoV-2” (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol and silica RNA capture matrices to recover 6-fold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, both suitable as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and could be performed in approximately 3 hours. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic., ART

Plasmodium vivax is a world-threatening human malaria parasite, whose biology remains elusive. The unavailability of in vitro culture, and the difficulties in getting a high number of pure parasites makes RNA isolation in quantity and quality a challenge. Here, a methodological outline for RNA-seq from P. vivax isolates with low parasitemia is presented, combining parasite maturation and enrichment with efficient RNA extraction, yielding ~ 100 pg.µL−1 of RNA, suitable for SMART-Seq Ultra-Low Input RNA library and Illumina sequencing. Unbiased coding transcriptome of ~ 4 M reads was achieved for four patient isolates with ~ 51% of transcripts mapped to the P. vivax P01 reference genome, presenting heterogeneous profiles of expression among individual isolates. Amongst the most transcribed genes in all isolates, a parasite-staged mixed repertoire of conserved parasite metabolic, membrane and exported proteins was observed. Still, a quarter of transcribed genes remain functionally uncharacterized. In parallel, a P. falciparum Brazilian isolate was also analyzed and 57% of its transcripts mapped against IT genome. Comparison of transcriptomes of the two species revealed a common trophozoite-staged expression profile, with several homologous genes being expressed. Collectively, these results will positively impact vivax research improving knowledge of P. vivax biology.

Rhizomania is a grave disease affecting sugar beet (Beta vulgaris L.). It is caused by the Beet Necrotic Yellow Vein Virus (BNYVV), an RNA virus transmitted by the plasmodiophorid vector Polymyxa betae. Genetic resistance to the virus has been accomplished mostly using phenotype-genotype association studies. As yet, the most convenient method to ascertain plant resistance has been the quantification of viral titer in roots through the ELISA test. This method is particularly time-consuming and clashes with the necessities of modern plant breeding. Here, we propose an alternative and successful phenotyping method based on the automatic extraction of the viral RNA from sugar beet roots and its relative and absolute quantification by quantitative real-time PCR (qRT-PCR) and digital PCR (dPCR), respectively. Such a method enables an improved standardization of the study, as well as an accurate quantification of the virus also in those samples presenting low virus titer, with respect to the ELISA test. Supplementary Information The online version contains supplementary material available at 10.1007/s13337-021-00674-7.

Necrotic enteritis (NE) is an important enteric disease in poultry and has become a major concern in poultry production in the post-antibiotic era. The infection with NE can damage the intestinal mucosa of the birds leading to impaired health and, thus, productivity. To gain a better understanding of how NE impacts the gut function of infected broilers, global mRNA sequencing (RNA-seq) was performed in the jejunum tissue of NE challenged and non-challenged broilers to identify the pathways and genes affected by this disease. Briefly, to induce NE, birds in the challenge group were inoculated with 1 mL of Eimeria species on day 9 followed by 1 mL of approximately 108 CFU/mL of a NetB producing Clostridium perfringens on days 14 and 15. On day 16, 2 birds in each treatment were randomly selected and euthanized and the whole intestinal tract was evaluated for lesion scores. Duodenum tissue samples from one of the euthanized birds of each replicate (n = 4) was used for histology, and the jejunum tissue for RNA extraction. RNA-seq analysis was performed with an Illumina RNA HiSeq 2000 sequencer. The differentially expressed genes (DEG) were identified and functional analysis was performed in DAVID to find protein–protein interactions (PPI). At a false discovery rate threshold

BackgroundHigh-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount.MethodsPlant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis.ResultsThe study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols.ConclusionsThis study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

Hepatitis E virus (HEV) is a biological hazard that must be controlled and is a recognized etiological agent in viral hepatitis. This is a zoonotic virus and can be transmitted through the fecal-oral route. The pig is an important reservoir host of HEV, and is a source of contamination for the consumer after the consumption of raw or undercooked pork products. When detected, the most prevalent genotype of HEV in China is genotype 4 (denoted as HEV-4). To ensure the safety of this food of animal origin, we undertook a survey of HEV contamination in pig livers and pork samples available for sale, in retail outlets in selected cities in China. Viral RNA was purified from samples collected by lysing in Trizol followed by purification using trichloromethane and virus RNA extract kit. An additional step was applied to improve the recovery rate by adding RNase OUT when extracting virus RNA from pig livers, and the RNA productions were washed in 75% (v/v) ethanol to remove inhibitors. In total, 158 pig livers and 80 pork samples were procured and analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After purification of total RNA from all samples taken and analyzed by RT-qPCR, a single pig liver was positive by this method for HEV. The positive rate was calculated as 0.63%. In this study, a single positive sample was detected. Considering the dietary habits of Chinese people, pork is a popular food that on occasion may be contaminated with HEV, thereby posing a threat to consumer health. Ongoing surveillance is required to assess the risk to human health arising from HEV-contaminated pork being offered for sale, at retail outlets, especially in the areas of China where pig production is practiced.

The objective of this study was to compare the transcription of gene markers for gastrointestinal (GI) epithelial cells, including fatty acid binding protein 2 (FABP2) and cytokeratin 8 (KRT8), and tight junction complex genes (TJP1, CLDN1, CLDN4) in fecal RNA against several GI tract tissue sections in dairy calves. Eight healthy Jersey calves were euthanized at 5 wk of age, and postmortem samples were collected from rumen, duodenum, jejunum, ileum, large intestine, cecum, and feces for total RNA isolation. Tissues and fecal samples were immediately frozen in liquid nitrogen until RNA isolation. A real-time quantitative PCR analysis was performed using a single standard curve composited of equal amounts of all samples, including cDNA from fecal and GI tract tissues. The mRNA expression of the tight junctions TJP1, CLDN1, and CLDN4 was greater in fecal RNA compared with lower GI tract tissues (i.e., duodenum, jejunum, ileum, large intestine, and cecum). Similar to fecal RNA, rumen tissue had greater expression of tight junctions CLDN1 and CLDN4 than lower GI tract tissues. Similarly, rumen tissue had greater expression of TPJ1 than all lower GI tract tissues except duodenum. The expression of TJP1 and CLDN4 was greater in fecal RNA than in rumen tissue; in contrast, CLDN1 mRNA expression was greater in rumen tissue than in the fecal RNA. The expression of FABP2 was greater in duodenum in comparison to all tissue except ileum. The mRNA expression of FABP2 in fecal samples was similar to jejunum and ileum. The expression of KRT8 in fecal samples was similar to duodenum, large intestine, and cecum. The fecal RNA had a greater expression of KRT8 in comparison to jejunum and ileum. The rumen tissue had the lowest mRNA expression of KRT8. The expression levels of FABP2, KRT8, and tight junction genes observed in fecal transcripts suggest that a considerable amount of RNA derived from GI tract epithelial cells can be detected in fecal RNA, which is in agreement with previous data in neonatal dairy calves and other biological models including humans, rodents, and primates. The greater expression of tight junctions in fecal RNA in comparison to sections of the low GI remains to be understood, and due to the importance of tight junctions in GI physiology, further clarification of this effect is warranted. The similarities in mRNA expression of FABP2 and KRT8 between fecal RNA and intestinal sections add up to the accumulating evidence that fecal RNA can be used to investigate molecular alterations in the GI tract of neonatal dairy calves. Further research in this area should include high-throughput transcriptomic analysis via RNA-seq to uncover novel molecular markers for specific sections of the GI tract of neonates.

Salinity is one of the most limiting constraints of crop production. Nevertheless, highly-tolerant crop plants (e.g. ‘Guar’ Cyamopsis tetragonoloba) can thrive well under salinity conditions. RNA-Seq plays a central role in identifying genes involved in the response mechanisms to salt stress in plant species. High-quality RNA, free of genomic DNA is a critical determinant for success in subsequent downstream experiments to shed light on tolerance mechanisms. In this research, an inspection of two different total RNA isolation protocols was employed for preparing highly pure and intact RNAs with good yield from Guar tissue under both high salinity and control conditions. The extracted RNA appeared in all quality metrics to be of adequate quantity and quality for most downstream applications, including high-throughput transcriptome sequencing and expression analysis. However, the outcomes showed that the most widely used RNA quality measurement, the RNA Integrity Number (RIN), is a poor indicator with a weak correlation to the integrity of RNA from this plant. In contrast to human and animal cells, plants have various ribosomal RNA species. Consequently, whenever the RNA was separated on an agarose gel, several RNA bands did appear, which gives the false impression of being unable to analyze the results of the bioanalyzer integrity. Low RIN number RNA samples were further processed by RNA-sequencing RNA-seq experiments from guar. RNA-seq analysis via Illumina’s paired-end method was carried out to trace back and evaluate the extracted total RNA, resulting in high-quality data for subsequent stress tolerance research.

Background Accumulating evidence shows that microRNAs are aberrantly expressed and exert essential roles in the tumorigenesis and tumor progression of non‐small cell lung cancer (NSCLC). Methods The plasma miRNAs from five healthy donors and four NSCLC patients were profiled by miRNA microarray. The differentially expressed miRNAs from 154 primary NSCLC patients and 146 healthy donors were subjected to RNA isolation and verified by quantitative PCR (qPCR). Results The miRNA microarray analysis revealed that 40 differential miRNAs between NSCLC patients and healthy donors were selected. We found that the plasma miR‐1247‐5p, miR‐301b‐3p and miR‐105‐5p levels of patients were significantly higher than those of healthy controls. The receiver operating characteristic curve (ROC) analyses revealed higher area under the ROC curve (AUC) values and higher sensitivity/specificity of carcinoembryonic antigen (CEA) in combination with miR‐1247‐5p, miR‐301b‐3p, or miR‐105‐5p were superior to that of CEA alone. Conclusions High miR‐1247‐5p, miR‐301b‐3p and miR‐105‐5p expression have been demonstrated to accelerate tumorigenesis, and these three miRNAs in plasma act as novel biomarkers for the early diagnosis of NSCLC patients. Key points Plasma miR‐1247‐5p, miR‐301b‐3p and miR‐105‐5p act as novel biomarkers for early NSCLC and NSCLC., In summary, our results indicate that miR‐1247‐5p, miR‐301b‐3p, and miR‐105‐5p are candidate biomarkers of NSCLC, which perform even better when combined with CEA levels. These findings also prompted us to hypothesize that the early dysregulation of miR‐1247‐5p, miR‐301b‐3p, and miR‐105‐5p in the NSCLC disease process, and the requirement of only a small amount of plasma, render miR‐1247‐5p, miR‐301b‐3p, and miR‐105‐5p very suitable as early warning biomarkers for NSCLC.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

We recently investigated the presence of enteroviruses (EVs) in non-human primates (NHPs) in Northern Nigeria and documented the presence of EV-A76 of South-East Asian ancestry in an NHP. In this study, we go further to ask if we could also find EVs in NHPs indigenous to the forested South-south Nigeria. Fresh faecal samples were collected from the floor of 10 cages housing NHPs in Cross River Nigeria, re-suspended in PBS and subjected to RNA extraction, cDNA synthesis, PanEnt 5'-UTR and PanEnt VP1 PCR assays. None of the samples was positive for the PanEnt VP1 assay, but one sample was positive for PanEnt 5'-UTR PCR. This sample was subsequently inoculated into RD cell line, produced CPE and the isolate analysed by PCR assays, next-generation whole genome sequencing and passage in four different cell lines showing replication in two of them. Analysis of the complete genome of the isolate identified it as an Echovirus 11 (E11) and revealed a recombinant genomic structure. Phylogenetic analysis showed that the E11 NHP strain was related to human clinical isolates suggesting a zoonotic behaviour. We describe the first isolation and complete genome characterization of an E11 obtained from an NHP in Nigeria having zoonotic potential.