Amplification Of cDNA Research Articles

IFN-γ Induces Distinct Single Cell Transcriptomes in Clinical-Grade Mesenchymal Stromal Cells

INTRODUCTION: Bone marrow derived mesenchymal stromal cells (BMSC) have been widely used to treat or prevent acute graft-versus-host disease, however the variable efficacies reported between clinical trials suggests considerable heterogeneity between clinical-grade BMSC products. Preincubation of BMSC with IFN-γ may improve their immunosuppressive qualities, reduce the functional diversity of the cell product, and decrease the variability in the clinical results obtained. We aimed to identify the distinct BMSC transcriptome pattern induced by IFN-γ and explored BMSC heterogeneity by studying transcriptional diversity at the single cell level.METHODS: Human clinical grade BMSC (n=3) were produced at the GMP Clinical Cell Processing Laboratory in the Department of Transfusion Medicine, NIH under an IRB-approved protocol (NCT01071577). BMSC at passage 3, were cultured with or without IFN-γ (10 µg/mL) and were harvested after 12 hours. Single cell 3' digital gene expression profiling for BMSC from three paired conditions (IFN-γ +/-) were performed via the10xTM GemCodeTM Single Cell Platform (10x Genomics, Pleasanton, CA USA) at a targeted cell number of 5000 cells. Single cell RNA-seq library generations, GEM-RT, and cDNA amplification were performed according to manufacturer's instruction and libraries were sequenced on Illumina Hiseq 3000. The Cell Ranger Single-Cell Software was used for sample demultiplexing, barcode processing, single-cell gene counting, and cell clustering.RESULTS: BMSC samples were analyzed for single cell transcriptome at an average of 5188 cells per condition with a mean number of reads of 50,789 per cell and a median of 4,482 genes per cell. Cluster analysis of the six pooled samples revealed clinical grade BMSCs comprised a heterogenous population of at least 17 clusters. Mean count of unique molecular identifiers (UMI) confirmed that all clusters ubiquitously expressed BMSC phenotype markers (CD105, CD73, and CD90). Predominant clusters in control BMSC (clusters 2, 5, and 6) were enriched with distinct expression profiles, indicative of the diversity and heterogeneity of BMSC. Based on Gene Ontology gene set enrichment analyses, cluster 2 was enriched for genes involved in the extracellular matrix, endoplasmic reticulum lumen, and cell adhesion with high expression of IL1β, a proinflammatory cytokine. Clusters 5 and 6 made up populations enriched with a similar set of genes contributing to the mitotic cell cycle checkpoint system, protein serine-threonine kinase activity, and cell division suggesting proliferation potency. Cluster 6 was further characterized by genes involved in DNA replication and ATP binding. IFN-ɣ dramatically altered cluster arrangement, influencing the cell population and its function. For example, major clusters in control BMSC (clusters 2, 5, 6, and 16) significantly diminished in population size and, instead, new clusters 1, 9, and 15 appeared with IFN-ɣ stimulation (Figure). These IFN-ɣ inducible clusters were enriched for genes regulating chemotaxis, leukocyte migration, and MHC class I and II in addition to the type I interferon signaling pathway. Cluster 1 also expressed IDO1, a heme enzyme producing gene responsible for exhibiting immunosuppressive activity. Cluster 9 possessed distinctive features of genes involved in positive regulation of cAMP-mediated signaling and transport vesicle membranes, suggesting extracellular vesicle and exosome activity. Irrespective of IFN-ɣ stimulation, stagnant clusters enriched with G-protein coupled receptor signaling pathway genes remained, representing a vital population in stem cell self-renewal function.CONCLUSION: Single cell RNA-seq analysis of clinical grade BMSCs revealed a significant heterogeneous population composed of multiple clusters with distinct gene expression patterns. IFN-γ further modified the landscape of transcriptomes of BMSC at a single cell level. This transcriptome diversity may explain the differences in the quality of BMSC products for clinical application. Ongoing experiments include determining the dynamics of the population structure responsible for BMSC diversity and correlating the patterns of single cell transcriptome with the clinical potency of BMSC products. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of T Cell Receptors Specific to Antigens Presented By HLA-B5401 on IPS Cell-Derived Hematopoietic Stem Cells in a Patient with Acquired Aplastic Anemia Carrying B5401-Lacking Leukocytes

[Background] Cytotoxic T lymphocytes (CTLs) specific to hematopoietic stem cells (HSCs) are thought to play an important role in the development of acquired aplastic anemia (AA). The presence of HLA class I allele-lacking leukocytes as a result of 6pLOH or allelic mutations in about 25% of AA patients strongly suggests that CTLs specific to antigens presented by the specific HLA alleles are involved in the destruction of normal HSCs as well as in the escape of HLA-lacking HSCs. Establishing CTLs from these patients by culturing the patients' CD8+ cells with targeted HSCs may be useful for identifying autoantigens of AA. However, it is generally impossible to obtain sufficient numbers of HSCs from the patients for functional assays and in vitro stimulation of patient's T cells with HSCs may induce clonal expansion of CTLs irrelevant to pathogenesis of AA. To solve these problems, we generated iPS cells (iPSCs) from an untreated AA patient who possessed HLA-B5401-lacking leukocytes for inducing HSCs and attempted to identify cDNAs of T-cell receptor (TCR) α and β chains of predominant T cells in the patient's bone marrow (BM) using a single T-cell analysis, which may specifically recognize autologous HSCs. [Methods] iPSCs were generated from the peripheral blood mononuclear cells of an AA patient (56-year-old man), whose moderate AA later responded to cyclosporine therapy, using Yamanaka's method. The patient's monocytes accounted for 20% wild-type, 20% 6pLOH(+) and 60% B5401-lacking cells at the time of sampling. CD34+ cells were induced from these iPSCs and injected into the BM of C57BL/6.Rag2 mice harboring NOD-Sirpa (BRGS). CD8+ T cells in the BM of the patient were subjected to a single cell sorting followed by cDNA amplification of TCRVβ chains. TCRα chains of five T cells that showed the five most recurrent TCRVβs were determined, and the TCRβ cDNAs were retrovirally transfected to Jurkat cells that lacked TCRβ chain and were transfected with IFN-γ cDNA. Specific responses of each TCR transfectant to iPSC-derived CD34+ cells were determined using ELISA for detecting IFN-γ. [Results] Five iPSC clones, including wild type (n=2), 6pLOH(+) (n=1), and B5401-lacking (B5401[-]) (n=2) clones, that had different mutations in B*54:01 were generated. Three-week culture of the iPSCs in the presence of various growth factors produced hematopoietic cells consisting of 50% CD34+ cells of each phenotype, which were able to reconstitute of BRGS mice with 3.5% to 8.9% human CD45+ cells in the peripheral blood at 9-12 weeks after injection. When the four TCR transfectants were tested for their response to wild-type and B5401-lacking CD34+ cells, all four transfectants secreted higher amounts of IFN-γ in response to wild-type cells than to B5401(-) CD34+ cells. [Conclusions] HSCs with original genotypes capable of reconstituting hematopoiesis in immunodeficient mice were successfully induced from iPSCs that had been generated from an AA patient's monocytes. The TCR transfectants showing a specific response to B5401(+) wild-type CD34+ cells will be useful for identifying the ligand peptides presented by B5401. DisclosuresNo relevant conflicts of interest to declare.

Circle-to-circle amplification coupled with microfluidic affinity chromatography enrichment for in vitro molecular diagnostics of Zika fever and analysis of anti-flaviviral drug efficacy

Sensitive viral diagnostic methods are increasingly in demand to tackle emerging epidemics. The Zika virus (ZIKV) is particularly relevant in tropical resource limited settings (RLS) and is associated with intermittent epidemics such as the recent 2016 ZIKV outbreak in South America, wherein Zika fever was classified by WHO as a public health emergency of international concern. Thus, there is an urgent need for widespread Zika fever diagnostics and efficient drug therapies. ZIKV diagnostics are typically performed using RT-qPCR in centralized laboratories. While extremely sensitive, RT-qPCR requires rapid heating-cooling cycles, combined with continuous fluorescence measurements to allow quantification, implying high costs and limiting availability of molecular diagnostics in RLS. Here, we report isothermal amplification of ZIKV cDNA using padlock probes followed by two rounds of Rolling Circle Amplification (RCA), termed as circle-to-circle amplification (C2CA), combined with a microfluidic affinity chromatography enrichment (μACE) platform. This platform allowed the detection of <17 vRNA copies per reaction mixture, equivalent to ∼3 aM, showed a positive correlation with RT-qPCR in both average (r = 0.80) and discrete (r = 0.95) signal modes, and was validated for drug efficiency tests using in vitro infected peripheral blood mononuclear cells from 3 healthy donors. This performance shows significant promise towards highly sensitive, albeit simple and cost-effective point-of-care viral diagnostics.

Low bias multiple displacement amplification with confinement effect based on agarose gel.

Multiple displacement amplification (MDA) is a popular single-cell whole-genome amplification (WGA) technique that can greatly improve the amplification efficiency of single-cell genomes. However, there is an inherent problem that cannot be completely solved, that is, the amplification bias. We here propose an improved MDA method based on low melting agarose gel, named gelMDA. Firstly, the agarose gel and solution were characterized with SEM and fluorescent reagent. Then, we used gelMDA for cDNA amplification in library preparation of RNA-seq, and conventional MDA was used as a comparison. The sensitivity, efficiency of gelMDA, and amplification bias were evaluated with fluorescence curve, product yield, and the sequencing results. Finally, gelMDA was used for single-cell transcriptome sequencing. The results showed that the sensitivity and product yield of gelMDA were significantly higher than those of conventional MDA. A lower coefficient of variation (CV) and a higher reproducibility were obtained from gelMDA sequencing results. A region of 30μm in diameter was amplified from the tissue sections and successfully sequenced. In conclusion, gelMDA obtained higher amplification efficiency and lower amplification bias in the present study. It suggested the great potential in single-cell RNA amplification and sequencing.

Recovery of Synthetic Zika Virus Based on Rio-U1 Isolate Using a Genetically Stable Two Plasmid System and cDNA Amplification

In 2016, the world experienced the unprecedented Zika epidemic. The ZIKV emerged as a major human pathogen due to its association with the impairment of perinatal development and Guillain–Barré syndrome. The occurrence of these severe cases of Zika points to the significance of studies for understanding the molecular determinants of flavivirus pathogenesis. Reverse genetics is a powerful method for studying the replication and determinants of pathogenesis, virulence, and viral attenuation of flaviviruses, facilitating the design of vaccines and therapeutics. However, the main hurdle in the development of infectious clones is the instability of full-length cDNA in Escherichia coli. Here, we described the development of a genetically stable and efficient infectious clone based on the ZIKV Rio-U1 isolated in the 2016 epidemic in Brazil. The employed strategy consisted of cloning the viral cDNA genome into two stable plasmid subclones and obtaining a high-quality cDNA template with increment in DNA mass for in vitro transcription by PCR amplification. The strategy for developing a ZIKV infectious cDNA clone designed in this study was successful, yielding a replicative and efficient clone-derived virus with high similarities with its parental virus, Rio-U1, by comparison of the proliferation capacity in mammal and insect cells. The infection of AG129 immunocompromised mice caused identical mortality rates, with similar disease progression and morbidity in the animals infected with the parental and the cDNA-derived virus. Histopathological analyses of mouse brains infected with the parental and the cDNA-derived viruses revealed a similar pathogenesis degree. We observed meningoencephalitis, cellular pyknosis, and neutrophilic invasion adjacent to the choroid plexus and perivascular cuffs with the presence of neutrophils. The developed infectious clone will be a tool for genetic and functional studies in vitro and in vivo to understand viral infection and pathogenesis better.

SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification.

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

Environmentally friendly method of RNA isolation

The diversity of organisms, tissues and cells is so great that, to date, no universal method for RNA extraction from these biological materials exist. The RNA isolation technique with a mix of guanidine thiocyanate, phenol, and chloroform is most widely used. Extraction and purification of RNA methods using selling guanidinium-phenol (TRIzol)-based and silica-based column kits have limitations on toxicity, or RNA isolation, particularly for plants, and scaling. The agents' toxicity is particularly relevant when employing for mass analysis in practice while gaining RNA preparations during the pandemics, epizootics, and epiphytotic. In modern diagnostics of infections at the molecular level, powerful RT-PCR technology is used, which amplifies the detection of RNA pathogens by hundreds of millions of times. We proposed obtaining RNA samples from viruses, bacteria, and plants for the reverse transcription reactions with a subsequent amplification of cDNAs by the polymerase chain reaction using potent and nontoxic chaotropic agent ammonium trichloroacetate. The method works in the analytical and preparative range and can be useful in the case of extraordinary circumstances during mass infections. Potentially this method can be adapted for obtaining RNA samples ready for the RT-isothermal PCR in the field.

First report of pear chlorotic leaf spot-associated virus on Japanese and European pears in Japan and its detection from an eriophyid mite.

In May 2018, three leaf samples were collected from Japanese pear trees cv. "Hosui" that exhibited typical chlorotic spot symptoms (Supplementary Figure S1) in a germplasm nursery in Tsukuba, Ibaraki. Total RNA was prepared using the rapid CTAB method (Gambino et al. 2008) for high-throughput sequencing, as described by Kubota et al. (2020). In brief, after removing ribosomal RNAs, a library was constructed by fragmenting RNA, synthesizing cDNA, and polymerase chain reaction (PCR) amplification. Sequencing was performed using NovaSeq 6000 sequencer (Illumina, San Diego, CA, U.S.A.) with paired-end 150 nt reads. De novo assembly was performed using CLC Genomics Workbench 11.0 Software (Qiagen, Hilden, Germany), with a minimum length of 500 bp. A total of 36,017 contigs derived from 33,565,182 reads were obtained and subjected to BLASTX search against the GenBank sequence database as of January 2019. Viruses commonly found in stone fruits, i.e., apple stem pitting virus, apple green crinkle-associated virus, apricot latent virus (foveaviruses), and apple stem grooving virus (a capillovirus), were detected. In addition, five contigs with amino acid sequence homologies to P1-P4 of known emaraviruses and the P7 of High Plains wheat mosaic virus (Tatineni et al. 2014) were detected and designated as PEV-Jp. The complete nucleotide (nt) sequences of the five segments of PEV-Jp were determined by Sanger sequencing of cloned reverse transcription (RT)-PCR amplification products using the primers shown in Supplementary Table S1; the 5'- and 3'-terminal sequences were RACE verified (Takara Bio, Shiga, Japan). In pairwise comparisons, the complete RNA1 to RNA5 of PEV-Jp (LC554756-760) shared 90.7% to 98.7% nt identities with those of PCLSaV-CG1 (MK602177-181), indicating that PEV-Jp is an isolate of PCLSaV. Using newly designed segment-specific primers (Supplementary Table S1), 12 symptomatic Japanese pear trees cv. "Kosui" sampled in 2020 from the same nursery tested positive for PCLSaV by RT-PCR while 12 symptomless trees were negative for the virus. Similar chlorotic spots, sometimes accompany necrotic spots, were observed on European pear (Pyrus communis) cv. "Le Lectier." (Fig. S1F) in Niigata in 2019; PCLSaV was detected by RT-PCR in leaf tissue samples from symptomatic trees (n = 3/3) but not in symptomless trees (n = 0/2). No vector for PCLSaV has been identified (Liu et al. 2020) but acaricide sprays in the early spring are effective for preventing occurrence of chlorotic spots in pear orchards (Nakai et al. 2018). Since the infestations of Eriophyes chibaensis Kadono, an eriophyid mite often observed on the Japanese pear (Fig. S1G to S1I) (Kadono, 1981), has been associated with occurrences of the chlorotic spots (Shimizu et al. 2019), samples of E. chibaensis individuals were collected from PCLSaV-positive Japanese pear cvs. "Akizuki" and "Kosui"and P. communis cv. "Le Lectier." for total nucleic acid isolations via phenol-chloroform extraction, followed by quantitative RT-PCR (Supplementary Table S1). The expected RNA1 and RNA5 specific 150 bp products were detected from mite samples collected from Akizuki (n = 6/12), Kosui (n = 13/18), and Le Lectier (n = 6/8). The results indicate that E. chibaensis can ingest PCLSaV and may be a potential vector for the virus, although additional experiments are needed to demonstrate its vector competency. To our knowledge, this is the first report of PCLSaV in Japan and the first report of its detection in E. chibaensis.

Single-Cell Transcriptomic Analysis of Hematopoietic Cells.

Single-cell RNA sequencing (scRNA-Seq) allows the complete and unbiased analysis of the transcriptional state of an individual cell. In the past 5years, scRNA-Seq contributed to the progress of the hematology field, advancing our knowledge of both normal and malignant hematopoiesis. Different scRNA-Seq methods are available, all relying on the conversion of RNA to cDNA, followed by amplification of cDNA in order to obtain a sufficient amount of genetic material for sequencing. Currently available scRNA-Seq platforms can be broadly divided into two categories: droplet-based and plate-based. Each of these approaches has advantages and disadvantages that need to be considered when designing the experiment. Here, we describe detailed protocols of two of the most used methods for scRNA-Seq of hematopoietic cells: Smart-Seq2 (plate-based) and 10× Genomics (droplet-based).

First Report of Alfalfa mosaic virus in Chayote in Italy.

Chayote (Sechium edule (Jacq.) Sw.) is a vigorous perennial and climbing cucurbits, native to Mesoamerica, and cultivated for alimentary purposes in the American continent, Australia, New Zealand, South Europe, Asia and Africa. During spring 2019, some chayote plants showing bright yellow vein banding rings and lines were observed in a private garden in South Italy (Campania region). Symptoms coalesced in some leaves, covering almost the whole foliar area. Double-stranded RNAs were extracted from symptomatic leaves of a single chayote plant and reverse-transcribed, randomly amplified, and submitted to Illumina sequencing (Marais et al., 2018). Reads were assembled using CLC Genomics Workbench 11.1 (http://www.clcbio.com). Contigs were then annotated by Blastn and Blastx comparison with the Genbank database, which allowed the identification of eight contigs of between 380 and 980 nucleotides sharing significant identity with alfalfa mosaic virus (AMV) genomic RNAs. No other viral contigs were identified. Mapping of reads on AMV genomic RNAs identified 4,209 AMV reads (1.26% of total reads) and allowed the scaffolding of the contigs into three scaffolds corresponding to the three AMV genomic RNAs. To complete the sequence of the AMV chayote isolate genome (named See-1), primers were designed from the contig sequences and used to amplify RACE PCR products spanning the 5' and 3' terminal regions of the three genomic RNAs using the SMARTer™ RACE cDNA Amplification Kit (Clontech, China). All amplicons were cloned into the pGEM-T vector (Promega, USA) and sequenced (three clones for each amplicon) by Microsynth Seqlab (Microsynth AG, Switzerland). Finally, the complete genomic sequences of the three RNAs were assembled by MacVector 17.5 (MacVector Inc., USA). The RNA1, RNA2 and RNA3 of See-1 are 3,643, 2,593 and 2,037 nt respectively (GenBank accession Nos. MT093209 to MT093211), and share the highest nt sequence identity with the RNA1 and RNA3 of AMV isolate (HZ) from tobacco (99.5% for RNA1, HQ316635; 98.7% for RNA3, HQ316637) and with the RNA2 of isolate AMV-Gym from Gynostemma pentaphyllum (98.1%, MH332898), both from China. AMV isolate See-1 was classified as belonging to subgroup I based on the presence of a BamH I and two AvaII sites in the CP ORF (Parrella et al., 2000). Reverse transcription polymerase chain reaction, using primers targeting the CP gene (Parrella et al., 2000), confirmed AMV infection in three symptomatic cayote plants including that used for Illumina sequencing, with 100% of nt sequence identity of amplicons. Three plants each of Chenopodium amaranticolor, Nicotiana benthamiana and Solanum lycopersicon were mechanically inoculated with sap from isolate See-1 infected plant, leading to the appearance of typical AMV symptoms in all three hosts ten days post-inoculation (Jaspars & Bos, 1980). This note describes the first detection of AMV in cayote in Italy and, to the best of our knowledge, in the world. In some areas of Southern Italy, climatic conditions are favorable enough to allow chayote development in the wild. Further studies would be desirable to determine the distribution and incidence of AMV in chayote and to understand the possibility that this species may play a role in AMV epidemiology, representing a threat to other susceptible crops.

Study presence of COVID-19 (SARS-CoV-2) in the sweat of patients infected with Covid-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease, which started in Wuhan, Chin, has now become a public health challenge in most countries around the world. Proper preventive measures are necessary to prevent the spread of the virus to help control the pandemic. Because, SARS-CoV-2 is new, its transmission route has not been fully understood. In this study, we aimed to investigate the presence of SARS‐CoV‐2 in the sweat secretion of COVID‐19 patients. Sweat specimens of 25 COVID- 19 patients were collected and tested for SARS‐CoV‐2 RNA by Real‐time Polymerase Chain Reaction (RT-PCR) method. After RNA extraction and cDNA amplification, all samples were examined for the presence of ORF-1ab and N genes related to COVID-19. Results annotated by Realtime PCR machines software based on Dynamic algorithm. The results of this study showed the absence of SARS-CoV-2 in the sweat samples taken from the foreheads of infected people. Therefore, it can be concluded that the sweat of patients with COVID- 19 cannot transmit SARS-CoV-2. However they can be easily contaminated with other body liquids.

Deciphering the Early Mouse Embryo Transcriptome by Low-Input RNA-Seq.

Early preimplantation embryos are precious and scarce samples that contain limited numbers of cells, which can be problematic for quantitative gene expression analyses. Nonetheless, low-input genome-wide techniques coupled with cDNA amplification steps have become a gold standard for RNA profiling of as minimal as a single blastomere. Here, we describe a single-cell/single-embryo RNA sequencing (RNA-seq) method, from embryo collection to sample validation steps prior to DNA library preparation and sequencing. Key quality controls and external Spike-In normalization approaches are also detailed.

An easy and robust method for the isolation of high quality RNA from coconut tissues

Background: Coconut tissues consist of a complex network of polysaccharides, proteins, polyphenols, and lipids that can bind to nucleic acids and pose difficulty in isolation. Certainly, a vigorous method is required to isolate high quality and quantity of RNA from such tissues for the purpose of downstream experiments. In this paper, we discuss a newly developed method for the Isolation of RNA from Complex Matrices (IRCM) method from coconut tissues. Results: The method is robust, cheap, and efficient for the extraction of quality RNA in high quantities from the solid endosperm of stored and fresh coconut (150 μg/g FW with A260/280 = 1.89 and 247.5 μg/g FW with A260/280 = 1.91), coconut apple (263.8 μg/g FW with A260/280 = 1.97), and coconut bud (1052.5 μg/g FW with A260/280 = 2.00). The other well established methods, such as Method of RNA Isolation from Palm (MRIP), Cetyl Trimethyl Ammonium Bromide (CTAB), TRIZOL, and RNA plant kit failed to isolate quality RNA in appreciable quantities from the coconut tissues. Furthermore, the resultant RNA performed well in the downstream experiment, that is, RT-PCR for the production and amplification of cDNA. Conclusions: From the study, we concluded that the present method will play a vital role in the extraction of high quality RNA from complex matrices in a short time. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

High-Throughput Cellular RNA Sequencing (HiCAR-Seq): Cost-Effective, High-Throughput 3' mRNA-Seq Method Enabling Individual Sample Quality Control.

High-throughput screening is one of the pillars of drug development. Unbiased transcriptome profiling is now widely used for a deeper understanding of a drug's mechanisms of action, off target effects, and cytotoxicity. Although currently available high-throughput RNA-Seq (HT RNA-Seq) methods such as PLATE-Seq, DRUG-Seq, and BRB-Seq serve these purposes, the inherent nature of these methods does not allow sample-wise sequencing library quality control. Here, we describe an HTR method called High-throughput CellulAr RNA Sequencing (HiCAR-Seq). HiCAR-Seq was optimized to work directly on cultured cells (as little as 1,000 cells) or 10 ng of total RNA. HiCAR-Seq involves reverse transcription from cultured cells or total RNA using oligo-dT primers followed by the PCR amplification of full-length cDNAs using sample-specific barcode primers in individual plate wells. Amplification of cDNA from every sample can be verified using Bioanalyzer. This step not only reveals cDNA amplification but also provides greater precision for pooling equal concentrations of cDNA from different samples. A single pooled cDNA library is made suitable for sequencing on Illumina sequencers using a tagmentation kit. Because HiCAR-Seq targets a small region at the 3' of the mRNAs, as little as 3 to 4 million reads/sample are enough to infer changes in gene expression in human or mouse cells. We believe that HiCAR-Seq represents a robust and competitive addition to the existing set of transcriptome-based high-throughput screening methods. © 2020 Wiley Periodicals LLC. Basic Protocol 1: cDNA synthesis and barcoding/enrichment PCR Basic Protocol 2: Nextera tagmentation/amplification, quantification, and sequencing.

Apple Russet Ring and Apple Green Crinkle Diseases: Fulfillment of Koch's Postulates by Virome Analysis, Amplification of Full-Length cDNA of Viral Genomes, in vitro Transcription of Infectious Viral RNAs, and Reproduction of Symptoms on Fruits of Apple Trees Inoculated With Viral RNAs.

Apple russet ring and apple green crinkle are graft-transmitted diseases first reported more than 60 years ago, but at present, no association between a specific virus (variant) and the disease has been clearly demonstrated. In this study, we conducted the following series of experiments to identify the causal viruses (variants) of these apple diseases; (1) comprehensive analysis by next-generation sequencing of all viruses in each apple tree affected with russet ring or green crinkle disease, (2) amplification of full-length genomic cDNA of viruses using primers containing the T3 promoter and the in vitro transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts to both herbaceous and apple plants, (4) analysis of sequence variants of viruses present in infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple seedlings combined with the virus-induced flowering technology using the apple latent spherical virus vector to reproduce the symptom on the fruit as soon as possible, and (6) reproduction of symptoms on the fruits of apple trees inoculated with sequence variants and the re-isolation of each virus variant from apples showing fruit symptoms. The results showed that one of the sequence variants of the apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on the fruits of infected apple trees and that a sequence variant of the apple stem pitting virus probably causes green crinkle symptoms on an infected apple fruit. Thus, we were able to fulfill Koch’s postulates to prove the viral etiology of both the apple russet ring and green crinkle diseases. We also propose an experimental system that can prove whether a virus found in diseased tissues is the pathogen responsible for the diseases when the etiology is undetermined.

‘Seq &amp; Destroy’: the full genome sequencing of archived wild type and vaccine rinderpest virus isolates prior to their destruction

Rinderpest, a once much feared livestock disease, was declared eradicated in 2011, however virus-containing material is still held in laboratories worldwide. Prior to the destruction of our institute’s stocks, we determined the full genome sequence of the distinct samples of rinderpest virus (RPV) in our repository. This data would decipher the historical epidemiology of RPV and allow for recovery of the virus should the need arise. For each sample (n=123), sequencing libraries were prepared using either transposon-based fragmentation of cDNA (Nextera XT DNA Library Prep kit) or single primer isothermal amplification (Trio RNA-Seq kit) and sequenced on the Illumina MiSeq. Regions of low or no coverage were re-sequenced using a Sanger sequencing approach. Examination of the sequences of RPV isolates has shown that the African isolates form a single disparate clade, rather than two separate clades as was previously believed. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the matrix and fusion protein coding sequences, and a group of African isolates where translation of the fusion protein begins from a non-standard start codon (AUA). In addition, the viruses that were force-passaged through alternate hosts such as rabbits or goats, appear to diverge from the clades that represent viruses which were maintained in the wild. Our unique set of sequence data will be invaluable for forensic epidemiology investigations in the event of an unforeseen outbreak and aid in the understanding of the evolution of related morbilliviruses.

A Highly Sensitive, Accurate, and Automated Single-Cell RNA Sequencing Platform with Digital Microfluidics

Single-cell RNA sequencing (scRNA-seq) is a powerful method in investigating single-cell heterogeneity to reveal rare cells, identify cell subpopulations, and construct a cell atlas. Conventional benchtop methods for scRNA-seq, including multistep operations, are labor intensive, reaction inefficient, contamination prone, and reagent consuming. Here we report a digital microfluidics-based single-cell RNA sequencing (digital-RNA-seq) for simple, efficient, and low-cost single-cell mRNA measurements. Digital-RNA-seq automates fluid handling as discrete droplets to sequentially perform protocols of scRNA-seq. To overcome the current problems of single-cell isolation in efficiency, integrity, selectivity, and flexibility, we propose a new strategy, passive dispensing method, relying on well-designed hydrophilic-hydrophobic microfeatures to rapidly generate single-cell subdroplets when a droplet of cell suspension is encountered. For sufficient cDNA generation and amplification, digital-RNA-seq uses nanoliter reaction volumes and hydrophobic reaction interfaces, achieving high sensitivity in gene detection. Additionally, the stable droplet handling and oil-closed reaction space featured in digital-RNA-seq ensure highly accurate measurement. We demonstrate the functionality of digital-RNA-seq by quantifying heterogeneity among single cells, where digital-RNA-seq shows excellent performance in rare transcript detection, cell type differentiation, and essential gene identification. With the advantages of automation, sensitivity, and accuracy, digital-RNA-seq represents a promising scRNA-seq platform for a wide variety of biological applications.

The processes of homeostasis, chemotaxis and organic and inorganic response are significantly up-regulated during short-term oral mucosal cells in vitro cultivation

Abstract Mucous membranes appear in various parts of the whole body performing similar functions. However, they differ based on where the mucosa is located. It functions as a barrier in such systems as: respiratory, urogenital and digestive . In this study we will be focusing strictly on the oral mucosa. Keratinocytes and fibroblasts, which mainly form the structure of the oral mucosa, are subjected to numerous factors. Being one of the million parts that build the animal organism, they are involved in various processes. In this study, we will try to confirm that in the in vitro culture of oral mucosa cells, the expression of our selected genes undergoes significant changes which are tied to such processes as: homeostasis, chemotaxis and organic/inorganic response of the organism. For this study, 20 pubertal crossbred Landrace gilts were used. After slaughter, samples of buccal pouch mucosa were obtained and transported to the laboratory. The excised tissue was prepared and processed due to protocols. The final pellet was resuspended in supplemented DMEM. Once the cultures attained 70–80% confluency, they were passaged. Total RNA from each pooled sample was subjected to two rounds of sense cDNA amplification. The cDNA was processed on microarrays. Analysis of the scanned arrays was performed. The files were imported into downstream data analysis software. The DAVID analysis showed that differently expressed genes belongs to 56 Gene ontology groups. In this paper we focused on “cellular divalent inorganic cation homeostasis”, “chemical homeostasis”, “chemotaxis”, “homeostatic process” and “response to organic substance” GO BP terms. These sets of genes were subjected to hierarchical clusterization procedure. In summary, the data we collected showed primarily changes in gene expression that occurred in the thirty-day cell culture of oral mucosa tissue. We assume that indicated genes could be new gene markers for studied processes. Running title: Homeostasis in oral mucosa cells

Overexpression of bone morphogenetic protein 3 (BMP-3) via raav-mediated gene transfer on the osteochondrogenic fate of human bone marrow-derived mesenchymal stem cells

Purpose: Injured adult articular cartilage is not capable of reliably regenerating an original hyaline structure. To improve the intrinsic healing activities of damaged cartilage, administration of genetically modified bone marrow-derived mesenchymal stem cells (MSCs) in cartilage lesions is a potent approach to improve cartilage repair. Here, we explored the potential benefits of overexpressing the bone morphogenetic protein 3 (BMP-3) [1] via clinically relevant recombinant adeno-associated virus (rAAV) vectors upon the commitment of human MSCs (hMSCs) as future therapeutic platforms for cartilage repair. Methods: rAAV vectors were packaged, purified, and titrated as previously described [2]. rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene and rAAV-hBMP-3 a human BMP-3 sequence (OriGene), both controlled by the CMV-IE promoter/enhancer [2]. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty, washed, and the pellets obtained were resuspended in DMEM, 10% FCS, 1% penicillin-streptomycin, and FGF-2 (10 ng/ml) for selection by adherence of progenitor cells and characterization of stem cell surface markers [2]. Cells (passage 2) were transduced with rAAV (rAAV-hBMP-3 or rAAV-lacZ: 40 μl each vector) or let untreated and kept either in monolayer cultures in DMEM, 10% FBS or as high-density aggregate cultures (2 x 105 cells) in chondrogenic medium (DMEM high glucose, ITS+ Premix, 1 mM pyruvate, 37.5 μg/ml ascorbate 2-phosphate, 10-7 M dexamethasone, 10 ng/ml TGF-β) for up to 21 days. Expression of BMP-3 was monitored by immunohistochemistry or by ELISA (R&D Systems) [2]. Histological analyses were performed on fixed monolayer cultures (ALP staining) or on paraffin-embedded sections of the aggregate cultures (5 μμ) (toluidine blue staining) [2]. Total RNA was extracted using the RNeasy Protect Mini kit (Qiagen) and reverse transcription was carried out with the 1st Strand cDNA Synthesis kit (Qiagen). cDNA amplification was performed via SYBR Green real-time RT-PCR [2]. Ct values were obtained for each target gene (SOX9, COL10A1) and GAPDH as a control for normalization, and fold inductions (relative to control samples) were measured using the 2-ΔΔCt method [2]. Each condition was performed in duplicate in three independent experiments. A t-test was employed with p < 0.05 considered statistically significant. Results: Successful BMP-3 overexpression was observed both in hMSC monolayer and aggregate cultures by application of rAAV-hBMP-3 relative to the control conditions (lack of vector delivery, reporter rAAV-lacZ gene transfer) over the whole period of evaluation (Fig. 1). Application of rAAV-hBMP-3 significantly increased the levels of cell proliferation in hMSC monolayer and aggregate cultures relative to the control conditions (up to 1.3- and 1.4 difference, respectively, p ≤ 0.001) (Table 1). Chondrogenic differentiation was evidenced by intense toluidine blue staining in hMSC aggregate cultures after 21 days, especially when the cells were treated with rAAV-hBMP-3 (Fig. 2). Similar results were noted regarding the osteogenic differentiation of the cells over time (Fig. 2). A real-time RT-PCR analysis revealed a reduced hypertrophic differentiation of the cells following rAAV-hBMP-3 transduction relative to the control treatments (up to 5-fold difference in COL10A1 expression, p ≤ 0.001), probably due to increased chondrogenic SOX9 gene expression levels (up to 8-fold difference, p ≤ 0.001) (Fig. 3). Conclusions: Gene transfer of BMP-3 via rAAV directs the osteochondrogenic commitment of hMSCs with a delay in hypertrophic differentiation. These Results demonstrate the possibility of modifying hMSCs by therapeutic BMP-3 gene transfer as a future, potential tool to enhance the repair processes in sites of osteochondral lesions. REFERENCES: [1] Wozney et al., Science 1988, 242:1528; [2] Venkatesan et al., Stem Cell Res Ther 2012, 3:22. ACKNOWLEDGMENTS: Work funded by the Deutsche Arthrose-Hilfe. The authors wish to thank Mary B. Goldring for helpful discussion.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)