Target mRNA Sequences Research Articles

Targeted mRNA sequencing helps to classify variants affecting splicing in hypertrophic cardiomyopathies

Targeted mRNA sequencing helps to classify variants affecting splicing in hypertrophic cardiomyopathies

Single cell multi-omic analysis identifies key genes differentially expressed in innate lymphoid cells from COVID-19 patients.

Innate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing. To gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection, we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression, transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls. We found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover, all ILC subsets displayed a significantly higher frequency of CD69-expressing cells, indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation, activation and homeostasis. In addition, differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC. Overall, this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection.

Expression Profiling of Adipogenic and Anti-Adipogenic MicroRNA Sequences following Methylmercury Exposure in Caenorhabditis elegans.

MicroRNA (miRNA) are important regulators of gene expression that respond not only to developmental and pathological cues, but also to environmental stimuli. Dyslipidemia is a hallmark of metabolic conditions and has been shown to significantly affect the expression of circulating miRNA sequences. Recently, our lab has shown that the environmental toxicant methylmercury (MeHg) causes dyslipidemia in the Caenorhabditis elegans model organism. While 10 and 20 μM MeHg increases the expression of adipogenic transcription factors and lipid-binding proteins in worms, there is limited information on how the toxicant affects the miRNA regulators of these genes. We hypothesized that MeHg would increase the expression of adipogenic miRNA sequences and/or decrease the expression of anti-adipogenic miRNA sequences. We further hypothesized that the target mRNA sequences for the miRNAs affected by MeHg would be consequently altered. We selected three potentially adipogenic (mir-34, mir-124, and mir-355) and three potentially anti-adipogenic (mir-240, mir-786, and let-7) miRNA sequences homologous to known human miRNA sequences altered in obesity, and quantified their levels 24 h and 48 h post MeHg treatment. At 24 h post exposure, MeHg significantly increased expression of both the adipogenic and anti-adipogenic miRNA sequences 1.5-3x above untreated control. By 48 h post exposure, only the adipogenic miRNA sequences were elevated, while the anti-adipogenic miRNA sequences were decreased by 50% compared to untreated control. These data suggest that there are developmental changes in miRNA expression over time following MeHg exposure. We next selected one target mRNA sequence for each miRNA sequence based on miRNA-mRNA relationships observed in humans. MeHg altered the gene expression of all the target genes assayed. Except for mir-34, all the tested miRNA-mRNA sequences showed a conserved relationship between nematode and humans. To determine whether the selected miRNA sequences were involved in lipid accumulation in response to MeHg, lipid storage was investigated in transgenic worm strains that lacked the specific miRNA strains. Of the six strains investigated, only the mir-124 and let-7 mutant worms had lipid storage levels that were statistically different from wild type, suggesting that these two sequences can be potential mediators of MeHg-induced lipid dysregulation.

The structural basis of mRNA recognition and binding by yeast pseudouridine synthase PUS1.

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or 'PUS' enzymes include those that rely on guide RNA molecules and others that function as 'stand-alone' enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 Å resolution. The enzyme recognizes and binds both strands in a helical RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

σ28-dependent small RNA regulation of flagella biosynthesis.

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.

Detection of long mRNA sequences by a Y-shaped DNA probe with three target-binding segments

Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped DNA probe with three target-binding segments was developed to detect tumor-related mRNA. This Y-shaped DNA probe (Y-probe) was assembled by six single DNA strands. Among these DNA strands, two DNA strands contained the split G-quadruplex sequence, and two DNA strands were modified with a pair of fluorophore and quencher, which were used to produce the detectable signal. In the presence of a long target mRNA sequence, target mRNA was hybridized with the three target-binding segments of the Y-probe, resulting in the increased fluorescence of G-quadruplex specific dye Thioflavin T and the decreased fluorescence of fluorophore, which could achieve the ratio detection of target mRNA. The Y-probe exhibited a low detection limit of 17.53 nM. Moreover, this probe showed high accuracy due to the benefits of three target-binding segments.

Increased miR-200c levels disrupt palatal fusion by affecting apoptosis, cell proliferation, and cell migration

The mammalian palate separates the oral and nasal cavities, facilitating proper feeding, respiration, and speech. Palatal shelves, composed of neural crest-derived mesenchyme and surrounding epithelium, are a pair of maxillary prominences contributing to this structure. Palatogenesis reaches completion upon the fusion of the midline epithelial seam (MES) following contact between medial edge epithelium (MEE) cells in the palatal shelves. This process entails numerous cellular and molecular occurrences, including apoptosis, cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT). MicroRNAs (miRs) are small, endogenous, non-coding RNAs derived from double-stranded hairpin precursors that regulate gene expression by binding to target mRNA sequences. Although miR-200c is a positive regulator of E-cadherin, its role in palatogenesis remains unclear. This study aims to explore the role of miR-200c in palate development. Before contact with palatal shelves, mir-200c was expressed in the MEE along with E-cadherin. After palatal shelf contact, miR-200c was present in the palatal epithelial lining and epithelial islands surrounding the fusion region but absent in the mesenchyme. The function of miR-200c was investigated by utilizing a lentiviral vector to facilitate overexpression. Ectopic expression of miR-200c resulted in E-cadherin upregulation, impaired dissolution of the MES, and reduced cell migration for palatal fusion. The findings imply that miR-200c is essential in palatal fusion as it governs E-cadherin expression, cell death, and cell migration, acting as a non-coding RNA. This study may contribute to clarifying the underlying molecular mechanisms in palate formation and provides insights into potential gene therapies for cleft palate.

Abstract 1356: The tumor immune microenvironment of recurrent nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is endemic in southern China and Southeast Asia. While primary tumors respond well to radiotherapy with or without chemotherapy, the management of recurrent/metastatic (RM) diseases is a challenge. What contributes to the refractoriness of RM NPC remains to be elucidated. In this study, we recruited patients who developed local recurrences or distal metastases. Gene expression profiles of tumor cells, tumor stroma and tumor-infiltrating immune cells in paired primary and RM NPC samples were obtained by targeted mRNA sequencing techniques. It was found that RM NPC, compared to their primary compartment, showed significant enrichment of immune cell-related gene signatures. This was associated with an altered immune cell composition, with enhanced T cell activation and exhaustion coupled with an M2-skewed macrophage population. Together, these data depict a more inflamed yet immunosuppressive microenvironment of RM NPC and highlight macrophages being a potential target to reinvigorate antitumoral immune responses. Citation Format: Jingyi Ma, Anne James, Jolene Poh, Magdalene Jahn May Chua, Qianhui Poh, Sharifah Azzah Binte Syed Mohamad Anis Alsagoff, Xinxiu Sam, Geksan Tan, Chekang Lim, Sathiyamoorthy Selvarajan, Tony Kiat Hon Lim, Jamie Chen Yee Mong, Chweeming Lim. The tumor immune microenvironment of recurrent nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1356.

Abstract P3-05-09: LAG3+ Tumor Infiltrating Lymphocytes Predict Outcome in Treatment Naïve Triple Negative Breast Carcinoma

Abstract Significance: Triple negative breast carcinoma (TNBC), characterized by lack of estrogen (ER) and progesterone receptors (PR) and absence of Her2/neu (Her2) receptor amplification, typically follows an aggressive course that includes high relapse rates, rapid progression, and poor outcome. Advances in treatment have been limited by molecular heterogeneity within TNBCs and lack of effective molecular targets. Although a subset of TNBC associated with tumor infiltrating lymphocytes (TILS) is more immunogenic than other breast cancers, response to immunotherapy has been variable and reported in only 10-40% of cases. Identification of prognostic immune biomarkers will identify novel therapeutic targets and facilitate appropriate patient selection for therapy. Aim: To analyze the immune gene expression profile in TNBC and identify a prognostic marker. Methods: Targeted mRNA sequencing was performed on formalin fixed paraffin embedded whole sections from 30 treatment naïve TNBC with TILs (15 cases with a favorable outcome (recurrence free overall survival (OS) ≥5yrs) and 15 with unfavorable outcome (never disease free or dead of disease in < 5yrs) using the HTG Edge Seq Assay (HTG Molecular Diagnostics, Inc. Tucson AZ) to analyze the differential expression of 1392 immune related genes in their Precision Immuno-Oncology Panel. The top 40 up- and down- regulated DE genes with adjusted p-value of < 0.001 were then validated against the TNBC mRNA profiles in The Cancer Genome Atlas (TCGA) database. LAG3 was one of four genes that showed significant differential expression in both data sets. We used immunohistochemistry (IHC) (Rabbit monoclonal AntiLAG3 antibody, Abcam plc.) to analyze LAG3 expression in whole sections from 57 consecutive TNBCs where tumor excision had been the first line treatment. All patients were female, age 29-89 years (median 55 yrs). The infiltrating ductal carcinomas varied from 0.6 to 9.0 cm (median 2.6 cm). All tumors were ER-, PR-, Her2- with a median Ki67 of 42%. 26 patients had metastatic carcinoma in 1-17 lymph nodes (median 2). Two patients had metastatic disease at presentation. Disease free survival (DFS) varied from 0 to 114.8 months (median 81.4 mos.), and overall survival (OS) varied from 7.6 to 114.8 months (median 81.5 mos.). LAG3 expression was categorized as negative (0), low (1+) and high (2+). The student t-test was used to correlate LAG3 expression with DFS and OS. Results: Differential expression analysis from our data set yielded 222 statistically significant differentially expressed genes. Validation against the TCGA TNBC data set revealed 4 common genes, one of which is LAG3. Only TILS showed LAG3 expression by IHC: 10 cases no expression, 17 low expression and 29 high expression. Median DFS and OS in TNBC with no LAG3 expression was 32.3 and 55.2 months, in TNBC with low expression 83.3 and 81.1 and those with high expression was 82 and 82.1 month. Median DFS and OS in LAG3 negative cases was significantly different from those in LAG3 positive cases (p=0.038, p=0.013) and between LAG3 0 and LAG3 2+ positive cases (p=0.002, p=0.0018). No difference in either median DFS or OS was seen when LAG3 1+ and LAG3 2+ cases were compared. Conclusions: 1. LAG3 is a prognostic marker for TNBCs. 2. LAG3 is expressed only on TILS and shows heterogenous expression. 3. TNBC with no LAG3 expression had a median survival of 32 months, suggesting they might benefit from more aggressive therapy than TNBC that express LAG3. 4. Confirmation of these results requires a larger TNBC cohort. Citation Format: Shikha Bose, Yizhou Wang, V Krishnan Ramanujan, Ann E. Walts. LAG3+ Tumor Infiltrating Lymphocytes Predict Outcome in Treatment Naïve Triple Negative Breast Carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-09.

Fe2O3@SiO2@Eu-MOF@Tb-MOF Core/Multishell Nanoparticles for the Fluorescence Sensing of Aspirin and Acute Myocardial Infarction Biomarkers

As a dangerous cardiovascular disease, acute myocardial infarction (AMI) has taken the lives of countless patients. Symptom onset of AMI is accompanied by a high amount of AMI biomarkers, including creatine kinase isoenzyme (CK-MB), troponin I (CTn I), and myoglobin (Mb). On the other hand, aspirin is an effective curing drug for AMI. Herein, we applied the layer-by-layer (LBL) synthesis strategy to form stratified magnetic core/multishell lanthanide metal–organic framework (Ln-MOF) Fe2O3@SiO2@Eu-MOF@Tb-MOF (MagMOF) nanoballs. Further, target mRNA sequence and antibodies (NH2-mRNA, anti-CTn, and anti-Mb) for AMI biomarkers are combined with MagMOF, respectively. They can be utilized as a hybrid sensing platform for the detection of CK-MB, Mb, and CTn I with high sensitivity (the limit of detection (LOD) for CK-MB is 174.98 U L–1, for CTn I is 0.16 mg L–1, and for Mb is 0.94 mg L–1). The detection ranges of MagMOF toward CK-MB, Mb, and CTn I in human serum have covered their maximum normal range in the human body, respectively. Further, MagMOF was applied in the ratiometric detection of aspirin with high sensitivity and low limit detection (LOD = 0.43 μM). As a result, MagMOF is the first example of realizing stable, susceptible, fast, and convenient detection of these AMI biomarkers and curing drug aspirin.

Long-term imaging of individual mRNA molecules in living cells

SummarySingle-cell imaging of individual mRNAs has revealed core mechanisms of the central dogma. However, most approaches require cell fixation or have limited sensitivity for live-cell applications. Here, we describe SunRISER (SunTag-based reporter for imaging signal-enriched mRNA), a computationally and experimentally optimized approach for unambiguous detection of single mRNA molecules in living cells. When viewed by epifluorescence microscopy, SunRISER-labeled mRNAs show strong signal to background and resistance to photobleaching, which together enable long-term mRNA imaging studies. SunRISER variants, using 8× and 10× stem-loop arrays, demonstrate effective mRNA detection while significantly reducing alterations to target mRNA sequences. We characterize SunRISER to observe mRNA inheritance during mitosis and find that stressors enhance diversity among post-mitotic sister cells. Taken together, SunRISER enables a glimpse into living cells to observe aspects of the central dogma and the role of mRNAs in rare and dynamical trafficking events.

MicroRNA-146a promotes proliferation, migration, and invasion of HepG2 via regulating FLAP

Abnormal expression of 5-Lipoxygenase Activating Protein (FLAP) has been detected in many tumor cells. MicroRNAs (miRNAs) negatively regulate gene expression post-transcriptionally by binding to the 3'–untranslated region (3'–UTR) of the target mRNA sequences and have been shown to be involved in various types of cancers. Herein, we aimed to demonstrate the expression of miR-146a and FLAP in human HCC tissues and liver cancer cell lines. We demonstrated that miR-146a expression is overexpressed, while FLAP protein and mRNA are suppressed in hepatocellular carcinoma tissues and HepG2 cells compared to para-carcinoma tissues and HL–7702 cells. Dual luciferase reporter gene assay showed that miR-146a-5p can directly target FLAP mRNA. Knockdown of miR-146a also resulted in increased FLAP expression of cancer cells. Additionally, miR-146a silencing or restoration of FLAP led to a reduction of HepG2 cell proliferation, cell cycle progression, migration, and invasion. This study showed that miR-146a has a stimulatory role in HepG2 cells and promotes HepG2 cell migration and invasion by targeting FLAP mRNA. Thus, miR-146a may be a tumor promoter and a potential therapeutic target for the treatment of HCC patients.

Exploring a peptide nucleic acid-based antisense approach for CD5 targeting in chronic lymphocytic leukemia.

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

Engineering miRNA features into siRNAs: Guide-strand bulges are compatible with gene repression

Synthetic siRNA guide strands are typically designed with perfect complementarity to the passenger strand and the target mRNA. We examined whether siRNAs with intentional guide-strand bulges are functional in vitro and in vivo. Importantly, this was done by systematic shortening of the passenger strand, evaluating identical 19-mer guide-strand sequences but forcing them into conformations with 1- to 4-nt bulges after annealing. We demonstrate that guide-strand bulges can be well tolerated at several positions of unmodified and modified siRNAs. Beyond that, we show that GalNAc-conjugated siRNAs with bulges at certain positions of the guide strand repress transthyretin in murine primary hepatocytes and in vivo in mice. In vivo, a GalNAc-conjugated siRNA with a 1-nt bulge at position 14 of the guide strand was as active as the perfectly complementary siRNA. Finally, in a luciferase reporter system, mRNA target sequences were systematically shortened so that RNA-induced silencing complex activity could only occur with a guide-strand bulge. Here, luciferase reporters were repressed when 1- and 2-nt deletions of the reporter were applied to the edges of the sequence. We conclude that some guide-strand bulges versus target transcript can result in target repression and therefore should be evaluated as off-target risks.

Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

RNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.

Model-Based Design of Synthetic Antisense RNA for Predictable Gene Repression.

Our enhanced understanding of RNA folding and function has increased the use of small RNA regulators. Among these RNA regulators, synthetic antisense RNA (asRNA) is designed to contain an RNA sequence complementary to the target mRNA sequence, and the formation of double-stranded RNA (dsRNA) facilitates gene repression due to dsRNA degradation or prevention of ribosome access to the mRNA. Despite the simple complementarity rule, however, predictably tunable repression has been challenging when synthetic asRNAs are used. Here, the protocol for model-based asRNA design is described. This model can predict synthetic asRNA-mediated repression efficiency using two parameters: the change in free energy of complex formation (ΔGCF) and percent mismatch of the target binding region (TBR). The model has been experimentally validated in both Gram-positive and Gram-negative bacteria as well as for target genes in both plasmids and chromosomes. These asRNAs can be created by simply replacing the TBR sequence with one that is complementary to the target mRNA sequence of interest. In principle, this protocol can be applied to design and build asRNAs for predictable gene repression in various contexts, including multiple target genes and organisms, making asRNAs predictably tunable regulators for broad applications.

Investigation of discordant sibling pairs from hereditary breast cancer families and analysis of a rare PMS1 variant

BackgroundIt is likely that additional genes for hereditary breast cancer can be identified using a discordant sib pair design. Using this design we identified individuals harboring a rare PMS1 c.605G>A variant previously predicted to result in loss of function. ObjectivesA family-based design and predictive algorithms were used to prioritize candidate variants possibly associated with an increased risk of hereditary breast cancer. Functional analyses were performed for one of the candidate variants, PMS1 c.605G>A. Methods1) 14 discordant sister-pairs from hereditary breast cancer families were identified. 2) Whole exome sequencing was performed and candidate risk variants identified. 3) A rare PMS variant was identified in 2 unrelated affected sisters but no unaffected siblings. 4) Functional analysis of this variant was carried out using targeted mRNA sequencing. ResultsGenotype-phenotype correlation did not demonstrate tracking of the variant with cancer in the family. Functional analysis revealed no difference in exon 6 incorporation, which was validated by analyzing PMS1 allele specific expression. ConclusionsThe PMS1 c.605G>A variant did not segregate with disease, and there was no variant-dependent impact on PMS1 exon 6 splicing, supporting this variant is likely benign. Functional analyses are imperative to understanding the clinical significance of predictive algorithms.

ZIKV Infection and miRNA Network in Pathogenesis and Immune Response.

Over the years, viral infections have caused severe illness in humans. Zika Virus (ZIKV) is a flavivirus transmitted by mosquito vectors that leads to notable neurological impairment, whose most dramatic impact is the Congenital ZIKV Syndrome (CZS). ZIKV targets neuronal precursor cells leading to apoptosis and further impairment of neuronal development, causing microcephaly, lissencephaly, ventriculomegaly, and calcifications. Several regulators of biological processes are involved in CZS development, and in this context, microRNAs (miRNAs) seem to have a fundamental role. miRNAs are important regulators of protein translation, as they form the RISC silencing complex and interact with complementary mRNA target sequences to further post-transcriptional repression. In this context, little is known about their participation in the pathogenesis of viral infections. In this review, we discuss how miRNAs could relate to ZIKV and other flavivirus infections.

FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells’ growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2.

The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.