Total RNA Samples Research Articles

Mycovirus Hunting Revealed the Presence of Diverse Viruses in a Single Isolate of the Phytopathogenic Fungus Diplodia seriata From Pakistan.

Diplodia seriata in the family Botryosphaeriaceae is a cosmopolitan phytopathogenic fungus and is responsible for causing cankers, fruit rot and leaf spots on economically important plants. In this study, we characterized the virome of a single Pakistani strain (L3) of D. seriata. Several viral-like contig sequences were obtained via a previously conducted next-generation sequencing analysis. Multiple infection of the L3 strain by eight RNA mycoviruses was confirmed through RT-PCR using total RNA samples extracted from this strain; the entire genomes were determined via Sanger sequencing of RT-PCR and RACE clones. A BLAST search and phylogenetic analyses indicated that these eight mycoviruses belong to seven different viral families. Four identified mycoviruses belong to double-stranded RNA viral families, including Polymycoviridae, Chrysoviridae, Totiviridae and Partitiviridae, and the remaining four identified mycoviruses belong to single-stranded RNA viral families, i.e., Botourmiaviridae, and two previously proposed families “Ambiguiviridae” and “Splipalmiviridae”. Of the eight, five mycoviruses appear to represent new virus species. A morphological comparison of L3 and partially cured strain L3ht1 suggested that one or more of the three viruses belonging to Polymycoviridae, “Splipalmiviridae” and “Ambiguiviridae” are involved in the irregular colony phenotype of L3. To our knowledge, this is the first report of diverse virome characterization from D. seriata.

An approach to quantitate maternal transcripts localized in sea urchin egg cortex using RT-qPCR with accurate normalization.

The sea urchin egg cortex is a peripheral region of eggs comprising a cell membrane and adjacent cytoplasm, which contains actin and tubulin cytoskeleton, cortical granules and some proteins required for early development. Method for isolation of cortices from sea urchin eggs and early embryos was developed in 1970s. Since then, this method has been reliable tool to study protein localization and cytoskeletal organization in cortex of unfertilized eggs and embryos during first cleavages. This study was aimed to estimate the reliability of RT-qPCR to analyze levels of maternal transcripts that are localized in egg cortex. Firstly, we selected seven potential reference genes, 28S, Cycb, Ebr1, GAPDH, Hmg1, Smtnl1 and Ubb, the transcripts of which are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, ΔCt, geNorm and NormFinder) based on calculated level of stability in both eggs as well as isolated cortices. Our results showed that gene ranking differs in total RNA and mRNA samples, though Ubb is most suitable reference gene in both cases. To validate feasibility of comparative analysis of eggs and isolated egg cortices, we selected Daglb-2 as a gene of interest, which transcripts are potentially localized in cortex according to transcriptome analysis, and observed increased level of Daglb-2 in egg cortices by RT-qPCR. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to determine cortical association of transcripts in sea urchin eggs.

A Pilot Study of Whole-Blood Transcriptomic Analysis to Identify Genes Associated with Repetitive Low-Level Blast Exposure in Career Breachers.

Repetitive low-level blast exposure is one of the major occupational health concerns among US military service members and law enforcement. This study seeks to identify gene expression using microRNA and RNA sequencing in whole-blood samples from experienced breachers and unexposed controls. We performed experimental RNA sequencing using Illumina’s HiSeq 2500 Sequencing System, and microRNA analysis using NanoString Technology nCounter miRNA expression panel in whole-blood total RNA samples from 15 experienced breachers and 14 age-, sex-, and race-matched unexposed controls. We identified 10 significantly dysregulated genes between experienced breachers and unexposed controls, with FDR corrected <0.05: One upregulated gene, LINC00996 (long intergenic non-protein coding RNA 996); and nine downregulated genes, IGLV3-16 (immunoglobulin lambda variable 3-16), CD200 (CD200 molecule), LILRB5 (leukocyte immunoglobulin-like receptor B5), ZNF667-AS1 (ZNF667 antisense RNA 1), LMOD1 (leiomodin 1), CNTNAP2 (contactin-associated protein 2), EVPL (envoplakin), DPF3 (double PHD fingers 3), and IGHV4-34 (immunoglobulin heavy variable 4-34). The dysregulated gene expressions reported here have been associated with chronic inflammation and immune response, suggesting that these pathways may relate to the risk of lasting neurological symptoms following high exposures to blast over a career.

Electrochemical detection of zeptomolar miRNA using an RNA-triggered Cu2+ reduction method

The development of ultrasensitive, selective, simple, and rapid microRNA (miRNA) detection strategies has been crucial because of their use as probable biomarkers for diagnosing human diseases. We herein described a label- and wash-free electrochemical miRNA detection method with zeptomolar sensitivity, which relies on target miRNA-induced reduction of Cu2+ and consequential changes in electrochemical signals generated from the remaining Cu2+. Target miRNA was successfully identified with a detection limit of 33.2 zM based on this simple principle. The synergistic combination of miRNA recycling and Cu2+ reduction reactions contributed to this ultrasensitivity. Moreover, the developed electrochemical sensing method exhibited label- and wash-free detection of miRNA, showing potential applicability as a point-of-care testing system. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the total RNA samples extracted from various cancer cell lines. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.

Stability investigation of air-dried olive ribo nucleic acids for metavirome studies

BackgroundThe application of ribo nucleic acids for molecular studies requires high integrity and quality of extracted total RNA samples. In addition, the need to transfer RNA samples at room temperature without special treatments such as ice and liquid nitrogen storage according to international transport laws highlights the importance of low cost alternative methods such as RNA air-drying, lyophilisation and transportable agents. In this study, the quality and quantity of air-dried RNA samples from leaf, petiole and bark tissues of different olive genotypes using several RNA extraction methods were compared with lyophilized ground leaves and RNAlater-stored tissue samples before precipitation. The quality of RNA and prepared libraries were checked by several techniques including agarose and polyacrylamide gel electrophoresis, Agilent quality control, RT-PCR amplification of housekeeping and viral genes and high throughput sequencing.ResultsAlthough RNA value varied amongst cultivars, RNA extraction with TRIzol™ Reagent in fresh extractions and samples stored in RNAlater before RNA extraction resulted in 455.26 ng/µL and 63.46 ng/µL (mean value of cultivars) as the highest RNA concentration averages, respectively. RNA samples extracted by TRIzol™ Reagents and stored for a short term at – 80 °C before air-drying showed the third highest concentration (44.87 ng/µL). The synthesized cDNAs quality for PCR amplification of housekeeping genes (Rbc 1 and Nad 5) and partial genomes of Arabis mosaic virus and Cucumber mosaic virus showed satisfactory results in RNA samples extracted by TRIzol™ Reagents despite its variation amongst cultivars.ConclusionsConsidering the difficulties in the extraction of high quality and quantity RNA in olive for molecular analyses, this study demonstrated that RNA extraction method based on TRIzol™ Reagent can be considered for virobiome studies of both fresh and air-dried samples.

An optimized method to\xa0obtain high-quality RNA from different tissues in Lilium davidii var. unicolor

The high quality, yield and purity total RNA samples are essential for molecular experiments. However, harvesting high quality RNA in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, different RNA extraction methods, namely TRIzol method, the modified TRIzol method, Kit method and cetyltrimethylammonium bromide (CTAB) method were employed to obtain total RNA from different tissues in L. davidii var. unicolor. A Nano drop spectrophotometer and 1% agarose gel electrophoresis were used to detect the RNA quality and integrity. Compared with TRIzol, Kit and CTAB methods, the modified TRIzol method obtained higher RNA concentrations from different tissues and the A260/A280 ratios of RNA samples were ranged from 1.97 to 2.27. Thus, the modified TRIzol method was shown to be the most effective RNA extraction protocol in acquiring RNA with high concentrations. Furthermore, the RNA samples isolated by the modified TRIzol and Kit methods were intact, whereas different degrees of degradation happened within RNA samples isolated by the TRIzol and CTAB methods. In addition, the modified TRIzol method could also isolate high-quality RNA from other edible lily bulbs. Taken together, the modified TRIzol method is an efficient method for total RNA isolation from L. davidii var. unicolor.

Abstract P3-06-06: Pg5 promotes breast cancer metastasis by activatingpi3k/akt/mtor signaling pathways

Abstract Background: Breast cancer is the most common malignancy and the leading cause of cancer death among women. Glycoprotein V (GP V) is involved in thrombin-induced platelet activation and in other platelet responses. However, its roles and underlying mechanisms in the pathogenesis of breast cancer are still unclear. This study aimed at assessing GP V expression in breast cancer, its subsequent effects on cancer cell apoptosis, proliferation, invasion, and migration, and the underlying molecular mechanisms. Methods: GP V mRNA expression was analysed in total RNA samples obtained from tumor and adjacent normal tissue of 41 breast cancer patients and cell lines (MDA-MB-231, MCF-7 and MCF-10A). The correlation between the expression of GP V and clinicopathological features was analyzed by immunohistochemical (IHC) staining. Stable knockdown and overexpression MDA-MB-231 cell lines were generated by using the lentiviral system. Then, the cells were harvested for CCK-8 assay, flow cytometry, scratch healing experiments, and western blotting. Two samples each from the Stable knockdown and control groups were used for transcriptome sequencing and pathway analysis. Finally key molecules associated with the related signaling pathways were analyzed by RT-PCR and western blot. Results: The results showed that the mRNA (P&amp;lt;0.0001) and protein expression (P&amp;lt;0.001) of GP V in the breast cancer tissues was significantly increased compared with in the adjacent non-tumor tissues. The protein and mRNA expression of GP V were significantly higher in MDA-MB-231 and MCF-7cell lines compared with human normal breast epithelial cells (MCF-10A) (P&amp;lt;0.01). GP V expression was positively correlated with higher histological grade and advanced TNM stage. (P&amp;lt;0.05), but was not related to gender, age, menopausal status, tumor size, vascular invasion, ER, PR, Ki-67 and lymph node metastasis (P&amp;gt;0.05). CCK-8 assay showed that cell viability was enhanced after GP V upregulation compared to the control group (P&amp;lt;0.05). The in vitro scratch assay showed that the number of GP-V-overexpressing cells migrating to the scratch area was significantly higher than that of the control group at 24 h and 48 h, respectively (P&amp;lt;0.001). Flow cytometric analysis demonstrated there was no significant difference in cell cycle distribution and apoptosis. Differential gene expression analysis showed 13 significantly down- and 141 upregulated mRNAs. The mRNA expression levels of PI3K and AKT were significantly increased after GP V upregulation compared with those in the control group (P&amp;lt;0.001). N-cadherin, Vimentin and MMP2/9 were up-regulated after GP V upregulation compared with those in the control group (P&amp;lt;0.05).Conclusion: GP5 is highly expressed in breast cancer tissues and may play an important role as a cancer-promoting gene in breast cancer. The high expression of GP5 was significantly associated with higher nuclear grade, TNM stage and Her-2 negativity. GP5 can promote the proliferation, invasion, and metastasis of breast cancer cells. GP5 may promote the proliferation, invasion, and metastasis by activating PI3K/AKT signaling pathway to upregulate MET, and is expected to become a potential target for clinic diagnosis and treatment. Citation Format: Kui Xiang, Wang Yan, Hua Yanshan, Yi Xiaojia, Zhao Chunmei, Guo Minmin. Pg5 promotes breast cancer metastasis by activatingpi3k/akt/mtor signaling pathways [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-06.

Aquaporin (AQP) channels in the spiny dogfish, Squalus acanthias I: Characterization of AQP3 and AQP15 function and expression, and localization of the proteins in gill and spiral valve intestine.

Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6k bases and AQP15 had a mRNA band of 2.1k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21kDa. For AQP15 a band of 26kDa was seen in gill and kidney. Fainter bands at 28 and 24kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue 'flaps'. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue.

Risk Factors for Severity and Mortality in Adult Patients Confirmed with COVID-19 in Sierra Leone: A Retrospective Study.

Background:The coronavirus disease 2019 (COVID-19) is a highly infectious respiratory disease. There is no recommended antiviral treatment approved for COVID-19 in Sierra Leone, and supportive care and protection of vital organ function are performed for the patients. This study summarized the clinical characteristics, drug treatments, and risk factors for the severity and prognosis of COVID-19 in Sierra Leone to provide evidence for the treatment of COVID-19.Methods:Data of 180 adult COVID-19 patients from the 34th Military Hospital in Freetown Sierra Leone between March 31, 2020 and August 11, 2020 were retrospectively collected. Patients with severe and critically ill are classified in the severe group, while patients that presented asymptomatic, mild, and moderate disease were grouped in the non-severe group. The clinical and laboratory information was retrospectively collected to assess the risk factors and treatment strategies for severe COVID-19. Demographic information, travel history, clinical symptoms and signs, laboratory detection results, chest examination findings, therapeutics, and clinical outcomes were collected from each case file. Multivariate logistic analysis was adopted to identify the risk factors for deaths. Additionally, the clinical efficacy of dexamethasone treatment was investigated.Results:Seventy-six (42.22%) cases were confirmed with severe COVID-19, while 104 patients (57.78%) were divided into the non-severe group. Fever (56.67%, 102/180) and cough (50.00%, 90/180) were the common symptoms of COVID-19. The death rate was 18.89% (34/180), and severe pneumonia (44.12%, 15/34) and septic shock (23.53%, 8/34) represented the leading reasons for deaths. The older age population, a combination of hypertension and diabetes, the presence of pneumonia, and high levels of inflammatory markers were significantly associated with severity of COVID-19 development (P < 0.05 for all). Altered level of consciousness [odds ratio (OR) = 56.574, 95% confidence interval (CI) 5.645–566.940, P = 0.001], high levels of neutrophils (OR = 1.341, 95%CI 1.109–1.621, P = 0.002) and C-reactive protein (CRP) (OR = 1.014, 95%CI 1.003–1.025, P = 0.016) might be indicators for COVID-19 deaths. Dexamethasone treatment could reduce mortality [30.36% (17/56) vs. 50.00% (10/20)] among severe COVID-19 cases, but the results were not statistically significant (P > 0.05).Conclusions:The development and prognosis of COVID-19 may be significantly correlated with consciousness status, and the levels of neutrophils and CRP.

Multiple stem-loop primers induced cascaded loop-mediated isothermal amplification for direct recognition and specific detection of circular RNAs.

Circular RNAs (circRNAs), as a novel class of endogenous noncoding RNAs, play important roles in many biological functions, such as acting as microRNA sponges and cancer biomarkers. Rapid and sensitive detection of circRNAs will be of great benefit to better understand their biological functions especially in clinical diagnosis using circRNAs as biomarkers. Herein, by using multiple stem-loop primers (SLPs) to specifically recognize the unique junction site of circRNAs, we demonstrate a multiple SLPs-induced cascaded loop-mediated isothermal amplification (LAMP) assay which has the ability to directly and specifically discriminate circRNAs from homologous linear RNAs without any linear RNA removal procedure. Taking advantage of the displacement activity of Bst DNA polymerase, the rationally designed SLPs can directly recognize the target circRNA and generate a large amount of double stem-loop DNAs which can initiate a cascaded LAMP reaction with improved efficiency. The use of additional SLPs can accelerate the reaction rate; as a result, the LAMP reaction can be finished within 35 min which is much quicker than traditional LAMP. The proposed method can detect as low as 1 fM circRNA and has been successfully applied to the detection of circRNAs in total RNA samples extracted from cell lines.

Deregulation of ncRNA in Neurodegenerative Disease: Focus on circRNA, lncRNA and miRNA in Amyotrophic Lateral Sclerosis.

Parallel and massive sequencing of total RNA samples derived from different samples are possible thanks to the use of NGS (Next Generation Sequencing) technologies. This allowed characterizing the transcriptomic profile of both cell and tissue populations, increasing the knowledge of the molecular pathological processes of complex diseases, such as neurodegenerative diseases (NDs). Among the NDs, Amyotrophic Lateral Sclerosis (ALS) is caused by the progressive loss of motor neurons (MNs), and, to date, the diagnosis is often made by exclusion because there is no specific symptomatologic picture. For this reason, it is important to search for biomarkers that are clinically useful for carrying out a fast and accurate diagnosis of ALS. Thanks to various studies, it has been possible to propose several molecular mechanisms associated with the disease, some of which include the action of non-coding RNA, including circRNAs, miRNAs, and lncRNAs which will be discussed in the present review. The evidence analyzed in this review highlights the importance of conducting studies to better characterize the different ncRNAs in the disease to use them as possible diagnostic, prognostic, and/or predictive biomarkers of ALS and other NDs.

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

BackgroundSingle-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing.ResultsHere, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified.ConclusionsThe ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.

A sensitive electrochemical method for rapid detection of dengue virus by CRISPR/Cas13a-assisted catalytic hairpin assembly

Dengue fever caused by Dengue virus (DENV) infection has been widely popular, especially in tropical and subtropical areas. Rapid and sensitive diagnosis is the first priority for treatment of DENV infection. This work designed a signal amplification strategy for sensitive electrochemical detection of DENV by using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system for catalytic hairpin assembly on electrode surface. The presence of target RNA could activate the cleavage activity of the CRISPR/Cas13a system to release the blocker silenced swing arms, which then hybridized with hairpin 1 (H1) immobilized on electrode surface to expose the pre-locked toehold domain of H1 for the hybridization of ferrocene-labeled hairpin 2 (H2-Fc). Eventually, a large number of H2-Fc were captured to the electrode to produce amperometric signal for achieving signal amplification. This method showed a linear detection range from 5 fM to 50 nM with a detection limit of 0.78 fM. The proposed assay was successfully used to detect DENV type 1 in total RNA sample extracted, indicating great potential for application in early clinical diagnostic.

335 DIFFERENTIALLY EXPRESSED MICRORNAS AND PREDICTIVE TARGET GENES IN BASALOID SQUAMOUS CELL CARCINOMA OF THE ESOPHAGUS

Abstract Background/Aim: Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide. Esophageal squamous cell carcinoma (ESCC) is considered as one of the most aggressive carcinomas of the gastrointestinal tract. Basaloid squamous cell carcinoma of the esophagus (BSCCE) is reported to have a poorer prognosis compared to conventional ESCC. The current study aimed to elucidate molecular differences between BSCCE and ESCC, using miRNA profiling and predictive target gene searching. Methods Materials and Methods: Four BSCCE and 94 ESCC patients who underwent esophagectomy were selected for this study. Cell lines were used for target gene validation. Total RNA samples, extracted from formalin-fixed paraffin-embedded blocks, were used for microarray profiling and validation of the miRNAs, selecting the candidate target genes, and elucidating their clinicopathological features. Furthermore, total RNA samples, extracted from miRNA mimic- and inhibitor-transfected cells in cell line experiments, were used for target gene validation. Both miRNA and mRNA quantifications were performed by quantitative reverse transcription-polymerase chain reaction. Results The microarray analysis revealed seven highly expressed miRNAs (miR-205-5p, −4732-5p, −1246, −3687, −3175, −6087, and − 1587) in the BSCCE patients when compared with control. We selected miR-4732-5p and − 3687 for the validation study, and target gene investigations were conducted for miR-3687 ultimately. Several candidates were selected after searching for the target genes via TargetScan and in the literature. Through a pilot and a validation study, progesterone receptor membrane component 2 (PGRMC2) was identified as a target gene. Further investigations revealed that PGRMC2 was associated with tumor size clinicopathologically. Conclusion miR-3687 may constitute a candidate marker of aggressiveness in BSCCE, and PGRMC2 is one of its target genes. Moreover, the gene may play a role in cell proliferation and local progression. Although the current study included only a small number of samples, this is the first report regarding differentially expressed miRNAs and predictive target genes in BSCCE patients.

Virus-Targeted Transcriptomic Analyses Implicate Ranaviral Interaction with Host Interferon Response in Frog Virus 3-Infected Frog Tissues.

Ranaviruses (Iridoviridae), including Frog Virus 3 (FV3), are large dsDNA viruses that cause devastating infections globally in amphibians, fish, and reptiles, and contribute to catastrophic amphibian declines. FV3’s large genome (~105 kb) contains at least 98 putative open reading frames (ORFs) as annotated in its reference genome. Previous studies have classified these coding genes into temporal classes as immediate early, delayed early, and late viral transcripts based on their sequential expression during FV3 infection. To establish a high-throughput characterization of ranaviral gene expression at the genome scale, we performed a whole transcriptomic analysis (RNA-Seq) using total RNA samples containing both viral and cellular transcripts from FV3-infected Xenopus laevis adult tissues using two FV3 strains, a wild type (FV3-WT) and an ORF64R-deleted recombinant (FV3-∆64R). In samples from the infected intestine, liver, spleen, lung, and especially kidney, an FV3-targeted transcriptomic analysis mapped reads spanning the full-genome coverage at ~10× depth on both positive and negative strands. By contrast, reads were only mapped to partial genomic regions in samples from the infected thymus, skin, and muscle. Extensive analyses validated the expression of almost all of the 98 annotated ORFs and profiled their differential expression in a tissue-, virus-, and temporal class-dependent manner. Further studies identified several putative ORFs that encode hypothetical proteins containing viral mimicking conserved domains found in host interferon (IFN) regulatory factors (IRFs) and IFN receptors. This study provides the first comprehensive genome-wide viral transcriptome profiling during infection and across multiple amphibian host tissues that will serve as an instrumental reference. Our findings imply that Ranaviruses like FV3 have acquired previously unknown molecular mimics, interfering with host IFN signaling during evolution.

Targeted Transcriptomics of Frog Virus 3 in Infected Frog Tissues Reveal Non-Coding Regulatory Elements and microRNAs in the Ranaviral Genome and Their Potential Interaction with Host Immune Response.

BackgroundFrog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae. Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions.ResultsUsing a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis-regulatory elements (CREs) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3’-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs).ConclusionsUsing the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response.

MelRNA-seq for Expression Analysis of SINE RNAs and Other Medium-Length Non-Coding RNAs

BackgroundSmall interspersed elements (SINEs) are transcribed by RNA polymerase III (Pol III) to produce RNAs typically 100–500 nucleotides in length. Although their RNA abundance can be evaluated by Northern blotting and primer extension, the nature (sequence, exact length, and genomic origin) of these RNAs cannot be revealed by these methods. Moreover, mRNA sequencing (mRNA-seq) is not able to distinguish bona fide SINE RNAs or SINE sequences present in longer transcripts.ResultsTo elucidate the abundance, source loci, and sequence nature of SINE RNAs, we established a deep sequencing method, designated as melRNA-seq (medium-length RNA-seq), which can determine whole-length RNA sequences. Total RNA samples were treated with 5′ pyrophosphohydrolase (RppH), which allowed ligation of an RNA adaptor to the 5′ end of intact SINE RNAs. Similarly, another adaptor was ligated to the 3′ end, followed by reverse transcription, PCR amplification, size selection, and single-end deep sequencing. The analysis of two biological replicates of RNAs from mouse spermatogonia showed high reproducibility of SINE expression data both at family and locus levels.ConclusionsThis new method can be used for quantification and detailed sequence analysis of medium-length non-coding RNAs, such as rRNA, snRNA, tRNAs, and SINE RNAs. Further, its dynamic range is much wider than Northern blotting and primer extension.

An Easy, Cost-Effective, and Scalable Method to Deplete Human Ribosomal RNA for RNA-seq.

RNA sequencing (RNA-seq) is a powerful and increasingly prevalent method to characterize and quantify the transcriptome. Ribosomes are extremely abundant, however, and approximately 80% of total RNA is ribosomal RNA (rRNA). Therefore, to detect and quantify less abundant yet biologically important transcripts such as messenger RNA (mRNA) and long noncoding RNAs (lncRNA), it is essential to minimize the rRNA being sequenced. Although commercial methods exist to deplete rRNA from total RNA samples before sequencing, they are expensive and require specific amounts of input RNA, and the most commonly used kit is no longer available as a stand-alone product. Here, we present an optimized rRNA depletion protocol using RNase H and DNA oligonucleotides complementary to human rRNA transcripts. This protocol includes guidelines for DNA oligo preparation, RNA:DNA hybridization, RNase H cleavage and RNA cleanup, and benchmarking of rRNA depletion. The method is flexible because the user can include additional complementary DNA oligos directed against any abundant transcript in their particular system. Furthermore, the performance of this rRNA depletion approach is comparable to or better than that of commercial kits, at a fraction of the cost and across a wide range of input RNA amounts. © 2021 Wiley Periodicals LLC. Basic Protocol: Specific depletion of rRNA transcripts from human total RNA Support Protocol: Preparation of the rRNA depletion DNA oligonucleotide pool.

Plasma differentially expressed genes and bioinformatics analysis of workers occupationally exposed to mercury

Objective: To screen and identify plasma differentially expressed genes and related signal pathway by human gene expression profile array and fluorescent quantitative PCR. Methods: From September 2018 to October 2019, 291 workers from a Mercury-in-glass thermometer factory in Jiangsu Province were selected for an occupational health examination, a total of 60 persons were divided into two groups: high and low mercury exposure groups (30 persons in each group) . Plasma total RNA samples from the high exposure group and the low exposure group (10 cases each) were detected by gene expression microarray, and differentially expressed genes (DEGs) with fold change >2 were selected. DEGs were submitted to David and Metascape for gene function clustering, pathway and protein interaction network analysis. Finally, fluorescence quantitative PCR was performed to verify the changes in the expression levels of key DEGs in the high exposure group and the low exposure group (another 20 cases in each group) . Results: A total of 269 DEGs, of which 203 up regulated and 66 down regulated were identified in the differential expression analysis of gene expression microarray. Bioinformatics analysis suggested that, DEGs were involved in forebrain development, glial cell fate determinants of GO biological process and PID NF-KB, PTEN signal pathway. NFE2L1, SOX8, SOX6 and RNF2 (P<0.05) were confirmed down regulated in high level group by fluorescent quantitative PCR compared with the low level group (fold changes were 2.10, 11.52, 2.19, and 4.38 respectively) . Conclusion: The plasma NFE2L1, SOX8, SOX6 and RNF2 gene expressions are significantly altered in occupa tional high mercury exposure population. PTEN signaling pathway and fate of glia cells determines the biological process may be closely related to the body injury caused by mercury exposure.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.