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At least 19 recordsLinked to original sources
PubMed2026

Spatial Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Tissue by spRandom-seq.

The molecular pathogenesis of infectious diseases and cancer is orchestrated by nanoscale of host and microbial RNA transcripts within the tissue microenvironment. Nevertheless, spatially resolving the comprehensive transcriptional landscape within complex clinical tissues, like formalin-fixed paraffin-embedded (FFPE) specimens, still poses a formidable challenge. Here, we present spRandom-seq, a random primer-based spatial total RNA sequencing technology designed to spatially resolve complete transcriptomes from host, bacteria, and even nanoscale viruses in FFPE tissues. Capitalizing on the random primer design, our technology not only facilitated the discovery of specific lncRNAs and alternative splicing events in mouse brain and olfactory bulb, but also delineated pronounced spatial heterogeneity in clinical FFPE sections-across distinct tumor regions in breast cancer and microbial infection sites in Klebsiella pneumoniae-infected tissues. Importantly, integrated analysis of host and viral RNAs in FFPE samples from hepatitis B virus (HBV)‑positive hepatocellular carcinoma (HCC) demonstrated that complement and coagulation pathways were specifically activated across expansive HBV‑infected tumor areas, which also exhibited an increased burden of copy number variations (CNVs). Owing to its compatibility with existing spatial transcriptomics platforms and minimal operational complexity, spRandom-seq represents a practical and scalable approach for clinical pathology applications and infection diagnostics.

Paraffin Embedding↗
PubMed2025

Concurrent stimulation of diflufenican biodegradation and changes in the active microbiome in gravel revealed by Total RNA.

The use of slowly degraded pesticides poses a particular problem when these are applied to urban areas such as gravel paths. The urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. We, therefore, endeavored to stimulate the degradation of the pesticide diflufenican added to urban gravel microcosms amended with dry alfalfa to increase microbial activity. In the present study, alfalfa addition significantly increased the formation of diflufenican's primary metabolite, 2-[3-(trifluoromethyl)phenoxy]nicotinic acid (AE-B), indicating stimulated biotransformation. The concurrent changes of the active microbial communities within the gravel were explored using shotgun metatranscriptomic sequencing of ribosomal RNA and messenger RNA. Although bacterial taxa remained dominant (87.0%-98.5% relative abundance), the alfalfa treatment led to a 4-5-fold increase in eukaryotic groups, including fungi and microbial grazers. Several microbial taxa potentially involved in the degradation of complex carbon compounds and aromatic pollutants-including Bacteroidetes, Verrucomicrobia, Sordariomycetes, Mortierellales, Tremellales, Sphingopyxis, and Phenylobacterium-increased in relative abundance following alfalfa amendment. Functional gene profiling revealed elevated expression of genes related to microbial activity and biomass production. Genes with potential roles in the breakdown of complex carbon structures (e.g., xylanases/chitin deacetylases) and in the transformation of aromatic compounds (e.g., ring-cleaving dioxygenases) were revealed. We conclude that complex carbon amendments can enhance the microbial activity, promoting the biotransformation of diflufenican in urban gravel environments. These findings provide new insights into the interactions between microbial community dynamics, gene expression profiles, and pesticide biotransformation in non-agricultural matrices.IMPORTANCEPesticides used on urban areas, e.g., gravel paths, are likely to have different effects and fates than when these are used on agricultural soils. Hence, studies into the degradation of pesticides applied to urban matrices are needed. We have previously shown that metabolites of the persistent pesticide diflufenican are even more persistent in urban soils, and it has also previously been shown that these metabolites leach from gravel surfaces. The reasons behind this are that the urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. In the present study, we, therefore, endeavored to stimulate the degradation of the pesticide diflufenican added to urban gravel microcosms amended with dry alfalfa to increase microbial activity, concurrently studying the changes in the active microbiome by Total RNA-metatranscriptomics.

Biodegradation, Environmental↗
PubMed1979

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA.

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images

Animals↗
PubMed1978

[Isolation and characterization of beef thyroid giant RNA].

Total HMW-RNAs were prepared by three different methods (method with phenol, method with NaClO4, method without phenol using Ultrogel AcA 22 filtration). Giant RNAs were obtained in the void volume by filtration on Sepharose 2B. The giant RNAs/total HMW-RNA ratio is higher (6.77%) with the gel filtration method than with phenol or NaClO4 methods (1.41% and 1.00% respectively). The nucleotide composition of these RNAs is DNA-like and the sedimentation constants are approximately 70-100 S.

Animals↗
PubMed1979

RNA synthesis in permeable mouse ascites sarcoma cells.

A permeable cell system for studying RNA synthesis was established. Mouse ascites sarcoma cells were made permeable to nucleoside triphosphates and alpha-amanitin by treating with a hypotonic buffer. Separate determinations of endogenous RNA polymerase I, II and III activities in permeable cells were conducted using the different sensitivities of these enzymes to alpha-amanitin. The endogenous activity of RNA polymerase II under optimal conditions was one tenth of total RNA synthetic activity in isolated nuclei, and one third of that in permeable cells. The extremely low ratio of RNA polymerase II activity to total RNA synthetic activity in isolated nuclei was thought to be caused by increase of RNA polymerase I activity and decrease of RNA polymerase II activity. These and other results suggested that RNA synthesis in permeable cells reflects more precisely the in vivo state of RNA synthesis than thatin isolated nuclei. The permeable cell system will provide a useful method for studying the separate activities of RNA polymerases I, II and III in situ.

Amanitins↗
PubMed1979

Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis.

Sublethal doses of gamma radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrophoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena.

Gamma Rays↗
PubMed2026

Dual Aberrant Splicing Caused by an Apparently Missense CHD7 Variant, c.5273A>G (p.Asp1758Gly), in CHARGE Syndrome.

CHARGE syndrome is a rare congenital disorder primarily attributed to heterozygous pathogenic variants of the CHD7 gene. Most pathogenic CHD7 variants are loss-of-function (LoF) variants, whereas the interpretation of missense variants remains challenging in the absence of functional evidence for their pathogenicity. We report a female infant presenting with clinical features characteristic of CHARGE syndrome. Targeted sequencing identified a heterozygous CHD7 variant (NM_017780.4:c.5273A>G), initially annotated as a missense substitution p.Asp1758Gly. This variant has been previously reported and registered with conflicting pathogenicity classifications; however, its transcript-level consequences remain unclear. Long-PCR-based RNA sequencing of total RNA from peripheral blood mononuclear cells revealed two aberrant splicing patterns associated with the variant: a predominant transcript carrying a 28-bp deletion due to cryptic donor splice-site activation, and a minor transcript with partial intron 24 retention. Both transcripts were predicted to result in premature termination codons. These findings demonstrate that c.5273A>G functions as a LoF variant through dual aberrant splicing rather than a simple missense substitution. This case underscores the importance of RNA-level splicing analysis for the accurate interpretation and classification of CHD7 missense variants.

CHD7↗
PubMed2025

Scalable single-cell total RNA-seq reveals non-coding programs in immunity, infection, and brain development.

Non-coding RNAs represent a widespread and diverse layer of post-transcriptional regulation across cell types and states, yet much of their diversity remains uncharted at single-cell resolution. This gap stems from the limitations of widely used single-cell RNA-sequencing protocols, which focus on polyadenylated transcripts and miss many short or non-polyadenylated RNAs. Here, we adapted single-cell RNA-sequencing on the 10x Genomics platform to capture a broad complement of coding and non-coding RNAs-including miRNAs, tRNAs, lncRNAs, histone RNAs, and non-adenylated viral transcripts. This approach enabled the discovery of rich, dynamic non-coding RNA programs across immune cells, virally infected hepatocytes, and the developing human brain. In dengue virus-infected hepatocytes, we detect non-adenylated viral transcripts and distinguish active from transcriptionally quiescent infected states, each with distinct host regulatory signatures. In brain tissue, we identify biotype-specific, cell-type-restricted non-coding RNAs, including miRNAs whose expression anticorrelates with predicted targets, consistent with post-transcriptional regulatory relationships. We show that MIR137, one of the strongest GWAS loci associated with schizophrenia and intellectual disability, is expressed specifically in Cajal-Retzius cells, an early-born but transient population that guides subsequent cortical neuron migration. These findings demonstrate the importance of non-coding RNAs in defining cell identity and state, and show how expanded transcriptome coverage can reveal additional layers of gene control-now accessible through practical and scalable single-cell profiling.

Journal Article↗
PubMed2024

Transcriptome analysis of the diseased intervertebral disc tissue in patients with spinal tuberculosis.

OBJECTIVE: To investigate the differential expression genes (DEGs) in spinal tuberculosis using transcriptomics, with the aim of identifying novel therapeutic targets and prognostic indicators for the clinical management of spinal tuberculosis. METHODS: Patients who visited the Department of Orthopedics at the Second Hospital, Lanzhou University from January 2021 to May 2023 were enrolled. Based on the inclusion and exclusion criteria, there were 5 patients in the test group and 5 patients in the control group. Total RNA was extracted and paired-end sequencing was conducted on the sequencing platform. After processing the sequencing data with clean reads and annotating the reference genome, FPKM normalization and differential expression analysis were performed. The DEGs and long non-coding RNAs (LncRNAs) were analyzed for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment. The cis-regulation of differentially expressed mRNAs (DE mRNAs) by LncRNAs was predicted and analyzed to establish a co-expression network. RESULTS: This study identified 2366 DEGs, with 974 genes significantly upregulated and 1392 genes significantly downregulated. The upregulated genes are associated with cytokine-cytokine receptor interactions, tuberculosis, and TNF-α signaling pathways, primarily enriched in biological processes such as immunity and inflammation. The downregulated genes are related to muscle development, contraction, fungal defense response, and collagen metabolism processes. Analysis of LncRNAs from bone tuberculosis RNA-seq data detected a total of 3652 LncRNAs, with 356 significantly upregulated and 184 significantly downregulated. Further analysis identified 311 significantly different LncRNAs that could cis-regulate 777 target genes, enriched in pathways such as muscle contraction, inflammatory response, and immune response, closely related to bone tuberculosis. There are 51 genes enriched in the immune response pathway regulated by cis-acting LncRNAs. LncRNAs that regulate immune response-related genes, such as upregulated RP11-451G4.2, RP11-701P16.5, AC079767.4, AC017002.1, LINC01094, CTA-384D8.35, and AC092484.1, as well as downregulated RP11-2C24.7, may serve as potential prognostic and therapeutic targets. CONCLUSION: The DE mRNAs and LncRNAs in spinal tuberculosis are both associated with immune regulatory pathways. These pathways promote or inhibit the tuberculosis infection and development at the mechanistic level and play an important role in the process of tuberculosis transferring to bone tissue.

Humans↗
PubMed2026

Virome of the Russian Grapevine Germplasm: A Final Study and Summary.

Ampelographic collections play an important role in the conservation of grapevine genetic resources and therefore require continuous phytosanitary monitoring. In this study, the virome of grapevines from the Magarach ampelographic collection in Russia was analyzed using total RNA high-throughput sequencing. A total of twenty-seven grapevine viruses and four viroids were identified. Two viruses were characterized as putative novel species: (+) ssRNA grapevine umbra-like virus 5 (GULV-5) and the bipartite (+) ssRNA grapevine Magarach secovirus (GMSV), which, together with related viruses, may represent a novel genus within the family Secoviridae. Among the economically important viruses, the most prevalent were grapevine fanleaf virus (76%), grapevine leafroll-associated virus 1 (39%), and grapevine virus A (33%). Mixed infections involving two or three of these viruses were detected in 50% of the analyzed grapevines. Grapevine virus D was detected in Russia for the first time. Phylogenetic analysis of 222 assembled virus and viroid genome sequences revealed high genetic diversity. The obtained results were summarized and compared with previous virome studies conducted on four Russian ampelographic collections.

RNA-Seq↗
PubMed1977

Human placental lactogen mRNA and its structural genes during pregnancy: quantitation with a complementary DNA.

A complementary DNA (cDNA) strand was transcribed from human placental lactogen (hPL) mRNA. Based on alkaline sucrose gradient centrifugation, the size of the cDNA was about 8 S, which would represent at least 80% of the hPL mRNA. Previously we showed that four to five times more hPL was synthesized in cell-free extracts derived from term as compared to first trimester placentas. Hybridization of the cDNA with RNA derived from placental tissue revealed that there was about four times more hPL mRNA sequences in total RNA from term placenta than in a comparable quantity of total first trimester RNA. Only background hybridization was observed when the cDNA was incubated with RNA prepared from human kidney. To test if this differential accumulation of hPL mRNA was the result of an amplification of hPL genes, we hybridized the labeled cDNA with cellular DNA from first trimester and term placentas and with DNA isolated from human brain. In all cases, the amount of hPL sequences was approximately two copies per haploid genome. Thus, the enhanced synthesis of hPL mRNA appears to result from a transcriptional activation rather than an amplification of the hPL gene. The increase likely reflects placental differentiation in which the proportion of syncytial trophoblast increases at term.

DNA↗
PubMed1979

Increased nuclease activity in cells treated with pppA2'p5'A2'p5' A.

A series of 2'-5'-linked oligo(adenylic acid) triphosphate (2'-5' A) inhibitors of protein synthesis were described recently. These inhibitors are synthesized from ATP by an enzyme activated in interferon-treated cell extracts or rabbit reticulocyte lysates by double-stranded RNA. We show here that 2'-5' A is a potent inhibitor of protein synthesis in intact cells of different origin (human, monkey, hamster, and mouse). At a concentration of 10 nM (in AMP equivalents), protein synthesis is inhibited by 50-85%. There is also a secondary effect on the total RNA synthesis which becomes evident several hours after inhibition of protein synthesis. All of these effects, however, are transient and, after a recovery period, both RNA and protein synthesis resume rates comparable to the appropriate controls. A nuclease activity is detected in cells after treatment with 2'-5'A. The total polyadenylylated RNA is much reduced in comparison to that from untreated cells, and electrophoretic analysis in polyacrylamide slab gels provides evidence for its degradation. Similarly, there is an apparent degradation of ribosomal RNA. Consistent with these results, extracts from cells that had been treated with 2'-5'A manifest an enhanced nuclease activity in vitro on incubation with exogenous RNA. Here, we propose that, as in cell-free systems, the mechanism of action of 2'-5'A in intact cells involves activation of a nuclease. This activation is transient, but the nuclease remains sensitive to further activation by the inhibitor.

Adenine Nucleotides↗
PubMed2025

Analysis of Leishbuviridae from Trypanosomatids.

Over the last decade, considerable progress has been made in unraveling RNA virus diversity. This has contributed to our understanding of the evolution of these viruses, which include emerging zoonotic human pathogens. Current success has been greatly facilitated by the development of next-generation sequencing platforms instrumental for meta-transcriptomic studies. However, due to the rapid evolution of RNA viruses, there are numerous "blind spots" waiting to be explored; one of those is the RNA virome of unicellular eukaryotes. Here, we present the pipeline, which has been successfully used to characterize various types of RNA viruses, including Leishbuviridae (Bunyaviricetes, Hareavirales) in the parasitic flagellates of the family Trypanosomatidae. The pipeline relies on axenic in vitro cell culture and double-stranded RNA enrichment, followed by direct RNA-sequencing. A detailed procedure description starting from the initial total RNA preparation to the final assembly of the viral segments is provided.

High-Throughput Nucleotide Sequencing↗
PubMed1975

Biochemical approach to bovine leukemia.

Many bovine leukemic lymphocytes produce virus particles when kept in survival cultures in Eagle's Minimum Essential Medium supplemented with 20 percent of inactivated fetal calf serum. Virus particles equilibrate at a density of 1.16 g/ml in sucrose gradients and at a density of 1.12 g/ml in metrizamide gradients. Simultaneous detection tests show that a high molecular weight RNA-reverse transcriptase complex exists in these viruses. Hybridizations between total RNA from bovine leukemic lymphocytes and C-DNA prepared in various known RNA oncogenic viruses show that the virus associated with bovine leukosis is unrelated to RLV, SSV-1, MSV-Ki, FSV-Ga and FLV-Ri.

Animals↗
PubMed1979

[Effects of different concentrations of chloramphenicol on RNA synthesis in E. coli].

In the presence of 2 microgram/ml chloramphenicol the rate of total RNA synthesis increases 1,6-fold, while that of rRNA synthesis--2,5--3,0-fold. Other ribosomal antibiotics, e.g. oxytetracycline, neomycin and puromycin, as well as chloramphenicol stimulate the synthesis of RNA while not causing a complete inhibition of the protein synthesis. Intensive stimulation of the rate of rRNA synthesis with low concentrations of chloramphenicol may be due to a decrease of intracellular concentration of guanosine-5'-diphosphate, 3'-diphosphate, as well as to the maintenance of a sufficiently high level of protein synthesis under these conditions.

Anti-Bacterial Agents↗
PubMed1977

Separation of newly-synthesized RNA by organomercurial agarose affinity chromatography.

Mouse myeloma cells, MOPC-31C, were incubated in the presence of 2-thiouridine and newly-synthesized RNA which appeared to contain 2-thioUMP as a constituent was separated from preexisting RNA by affinity chromatography using organomercurial agarose as a support. Both pH and salt concentration greatly affected the specific adsorption of the newly-synthesized RNA on the column. Under optimal conditions the rate of adsorption of the newly-synthesized RNA on the column was proportional to the logarithmic concentration of 2-thiouridine in the culture medium. Furthermore, at a given concentration of 2-thiouridine in the medium, a shorter incubation period caused a reduction of the rate of RNA adsorption on the column. The molecular size distributions of both total RNA and the adsorbed fraction, synthesized during 30 min in the presence of 2-thiouridine, were similar to that of RNA synthesized in the absence of the drug.

Cell Line↗
PubMed2025

Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

Exoribonucleases↗
PubMed2025

SMART-RNA-Metavirome: a practical RNA metavirome platform compatible with high-throughput sequencing of both short and long reads.

BACKGROUND: The RNA virosphere's extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens, including co-infections. However, enriching low-abundance viral sequences in RNA metaviromics, particularly in the preparation of cDNA libraries and their compatibility with next-generation sequencing (NGS) and third-generation sequencing (TGS), remains challenging. Therefore, our objective is to develop and systematically assess a practical RNA metavirome methodology specifically tailored for the enrichment of low-abundance viral sequences within samples. METHODS: We developed the SMART-RNA-Metavirome platform, integrating SMART-9n library preparation with NGS and TGS technologies. Total RNA was extracted from two field-collected wild Aedes albopictus pools, along with one laboratory-infected Ae. albopictus pool harboring dengue virus (DENV). This RNA was subjected to reverse transcription using both this optimized protocol and random primer-based methods, followed by high-throughput sequencing on Illumina, Oxford Nanopore, and QitanTech Nanopore technologies. Welch's t-test was employed for comparative analysis of the subsequent RNA metavirome data, specifically to evaluate differences in viral species composition and abundance of viral reads between experimental groups. Furthermore, the effectiveness of this platform was systematically validated via RT-qPCR and SMART-RNA-Metavirome-based Oxford Nanopore sequencing across multiple sample types, including mosquito specimens from DENV-infected Ae. albopictus, serum samples from dengue patients and viral isolates of Japanese encephalitis virus (JEV) and Zika virus (ZIKV). RESULTS: The SMART-RNA-Metavirome platform has been systematically validated to excel in enriching the composition and diversity of the RNA virome (P = 0.04), providing sufficient coverage for the complete reconstruction of viral genomes. When employed in the detection of DENV-infected Ae. albopictus, clinical serum samples, and viral isolates of JEV and ZIKV, this technique exhibits a robust correlation with RT-qPCR (r2 > 0.95). Notably, it demonstrates exceptional sensitivity, ensuring sufficient coverage even in samples of DENV-infected Ae. albopictus with a Ct-value of 35.3, attaining an impressive 99.88% genome coverage. Furthermore, this platform possesses the capability to identify virus species and determine their serotypes. CONCLUSIONS: In our study, the SMART-RNA-Metavirome platform outperforms traditional methods, enriching RNA virome composition and diversity, enabling practical compatibility with both NGS and TGS technologies. It demonstrates significant proficiency in detecting both known and unknown arboviruses, even in low-titer samples such as those from wild mosquitoes and clinical sera. This platform facilitates comprehensive monitoring, risk assessment, and early warning of RNA virus transmissions, enhancing our understanding of RNA virome diversity and ecological patterns.

High-Throughput Nucleotide Sequencing↗