Enrichment Of mRNA Research Articles

Oxidative Stress Mediated by N6-Methyladenosine Methylation Contributes to High-Fat Diet Induced Male Reproductive Dysfunction.

To determine the mechanism of oxidative stress mediated by N6-methyladenosine (m6A) methylation contributing to high fat diet-induced reproductive dysfunction. In vivo, compared with those in the Control group, the sperm count and sperm motility decrease significantly; the testosterone, luteinizing hormone levels, hyaluronidase, acrosomal enzyme levels, and total antioxidant capacity decrease significantly; malondialdehyde increases significantly in the DIO and DIO-R groups. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), superoxide dismutase 1 (SOD1), and NAD(P)H quinone dehydrogenase 1 (NQO1) decreases significantly in the DIO and DIO-R groups; m6A levels in testis tissue in the DIO and DIO-R groups increase; the enrichment of m6A-modified Nrf2 mRNA in testis in the DIO group and DIO-R group increases significantly. Also the m6A regulatory proteins increase significantly in the DIO group and DIO-R group. In vitro, compared to palmitic acid treated cells, the reactive oxygen species (ROS) level significantly decreases in STM2457, S-Adenosylhomocysteine treated cells and YTHDC2, YTHDF2 gene silence cells; however, Nrf2 expression increases in all treated cells. In addition, m6A expression decreases. Oxidative stress mediates by methylation of m6A may contribute to high fat diet-induced male reproductive dysfunction.

Combined modelling of mRNA decay dynamics and single-molecule imaging in the Drosophila embryo uncovers a role for P-bodies in 5' to 3' degradation.

Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. Here, we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3' ends in P-bodies in the early embryo, consistent with 5' to 3' degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.

Transcripts of the Prostate Cancer-Associated Gene ANO7 Are Retained in the Nuclei of Prostatic Epithelial Cells.

Prostate cancer affects millions of men globally. The prostate cancer-associated gene ANO7 is downregulated in advanced prostate cancer, whereas benign tissue and low-grade cancer display varying expression levels. In this study, we assess the spatial correlation between ANO7 mRNA and protein using fluorescent in situ hybridization and immunohistochemistry for the detection of mRNA and protein in parallel sections of tissue microarrays prepared from radical prostatectomy samples. We show that ANO7 mRNA and protein expression correlate in prostate tissue. Furthermore, we show that ANO7 mRNA is enriched in the nuclei of the luminal cells at 89% in benign ducts and low-grade cancer, and at 78% in high-grade cancer. The nuclear enrichment of ANO7 mRNA was validated in prostate cancer cell lines 22Rv1 and MDA PCa 2b using droplet digital polymerase chain reaction (ddPCR) on RNA isolated from nuclear and cytoplasmic fractions of the cells. The nuclear enrichment of ANO7 mRNA was compared to the nuclearly-enriched lncRNA MALAT1, confirming the surprisingly high nuclear retention of ANO7 mRNA. ANO7 has been suggested to be used as a diagnostic marker and a target for immunotherapy, but a full comprehension of its role in prostate cancer progression is currently lacking. Our results contribute to a better understanding of the dynamics of ANO7 expression in prostatic tissue.

Single-cell transcriptomics and data analyses for prokaryotes-Past, present and future concepts.

Transcriptomics, or more specifically mRNA sequencing, is a powerful tool to study gene expression at the single-cell level (scRNA-seq) which enables new insights into a plethora of biological processes. While methods for single-cell RNA-seq in eukaryotes are well established, application to prokaryotes is still challenging. Reasons for that are rigid and diverse cell wall structures hampering lysis, the lack of polyadenylated transcripts impeding mRNA enrichment, and minute amounts of RNA requiring amplification steps before sequencing. Despite those obstacles, several promising scRNA-seq approaches for bacteria have been published recently, albeit difficulties in the experimental workflow and data processing and analysis remain. In particular, bias is often introduced by amplification which makes it difficult to distinguish between technical noise and biological variation. Future optimization of experimental procedures and data analysis algorithms are needed for the improvement of scRNA-seq but also to aid in the emergence of prokaryotic single-cell multi-omics. to help address 21st century challenges in the biotechnology and health sector.

Urinary cell mRNA profiling of kidney allograft recipients: Development of a portable protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy

BackgroundWe developed urinary cell mRNA profiling for noninvasive diagnosis of acute T cell mediated rejection (TCMR) and BK virus nephropathy (BKVN), two significant post-transplant complications. Our profiling protocol for the multicenter Clinical Trial of Transplantation-04 (CTOT-04) study consisted of centrifugation of urine to prepare cell pellets, washes, addition of an RNA preservative, storage at 800C and shipment in cold containers to our Gene Expression Monitoring (GEM) Core for RNA isolation and quantification of mRNA in RT-qPCR assays. To simplify profiling, we developed a filter-based protocol (ZFBP) that eliminated the need for centrifugation, RNA preservative, storage at 800C, and shipment in cold containers for mRNA profiling. Furthermore, we trained kidney allograft recipients to perform the filtration of urine at home using the filter and post the urinary cell lysate containing the RNA at ambient temperature to our GEM Core for profiling. Here, we report our refinement of ZFBP and investigation of its diagnostic performance characteristics. MethodsTotal RNA was isolated from kidney allograft biopsy-matched urines using a filter-based protocol complemented by a silica-membrane-based cartridge for mRNA enrichment, the Weill Cornell Hybrid Protocol (WCHP). Absolute copy numbers of CD3ε mRNA, CXCL10 mRNA, and 18S rRNA, components of the CTOT-04 three-gene TCMR diagnostic signature, and urinary cell BKV VP 1 mRNA copy number were measured using RT-qPCR assays. Mann-Whitney test, Fischer exact test, and receiver operating characteristic (ROC) curve analysis were used for data analyses. ResultsUrinary cell three-gene TCMR diagnostic signature scores in urines processed using the WCHP discriminated kidney allograft recipients with TCMR (12 TCMR biopsies from 11 patients) from those without TCMR or BKVN (29 No TCMR/No BKVN biopsies from 29 patients). The median (25th and 75th percentiles) score of the CTOT-04 three-gene TCMR diagnostic signature was −0.448 (−1.664, 0.204) in the TCMR group and − 2.542 (−3.267, −1.365) in the No TCMR/ No BKVN group (P = 0.0005, Mann-Whitney test). ROC curve analysis discriminated the TCMR group from the No TCMR/ No BKVN group; the area under the ROC curve (AUROC) was 0.84 (95% Confidence Intervals [CI], 0.69 to 0.98) (P < 0.001), and TCMR was diagnosed with a sensitivity of 67% (95% CI, 35 to 89) at a specificity of 86% (95% CI, 67 to 95) using the CTOT-04 validated cutpoint of −1.213 (P = 0.0016, Fisher exact test). BKV VP1 mRNA copy number in urines processed using the WCHP discriminated patients with BKVN (n = 7) from patients without TCMR or BKVN (n = 29) and the AUROC was 1.0 (95% CI, 1.00 to 1.00) (P < 0.0001) and BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at a specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5 × 108 BKV-VP1 mRNA copies per microgram of RNA (P < 0.0001, Fisher exact test). ConclusionUrine processed using the WCHP predicted TCMR and BKVN in kidney allograft recipients. WCHP represents not only a significant advance toward the portability of urinary cell mRNA profiling but also improved patient management by minimizing their visits for urine collection.

Antisense oligonucleotides targeting miR‐29b binding site increase translation of progranulin protein: potential therapeutic strategy for progranulin‐deficient frontotemporal dementia

AbstractBackground GRN mutations cause frontotemporal dementia (FTD) due to haploinsufficiency of progranulin. Several microRNAs (miRs), including miR‐29b, have been reported to negatively regulate progranulin protein levels. Here, we tested if antisense oligonucleotides (ASOs) – which are versatile modulators of target mRNA/protein levels – can be used to increase progranulin levels by sterically blocking the miR‐29b binding site.MethodWe designed 48 ASOs targeting the miR‐29b binding site in the 3’ UTR of the human GRN mRNA. We treated H4 neuroglioma cells and iPSC‐derived neurons with these ASOs and subsequently measured progranulin protein levels by western blot and ELISA. We performed further studies to determine the mechanism of action of these ASOs using ribosomal profiling, metabolic labeling, and FRET assays.ResultsWe identified 16 ASOs that increased progranulin protein levels in a dose‐dependent manner. Ribosomal profiling experiments revealed that cells treated with ASOs had marked enrichment in GRN mRNA in heavy polyribosome fractions, compared to cells treated with a scrambled control ASO, suggesting that the ASOs increase the rate of progranulin translation. Consistent with this, ASO treatment resulted in increased levels of newly synthesized progranulin protein. FRET‐based assays showed that ASOs can effectively compete miR‐29b from its binding site in the GRN 3’ UTR RNA under in vitro conditions.ConclusionsTogether, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This strategy may be therapeutically feasible for progranulin‐deficient FTD as well as other conditions of haploinsufficiency.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Long Non-Coding RNA Expression Profile Alteration Induced by Titanium Dioxide Nanoparticles in HepG2 Cells.

The liver is considered the major target organ affected by oral exposure to titanium dioxide nanoparticles (TiO2 NPs), but the mechanism of hepatotoxicity is not fully understood. This study investigated the effect of TiO2 NPs on the expression profile of long non-coding RNA (lncRNA) in hepatocytes and tried to understand the potential mechanism of hepatotoxicity through bioinformatics analysis. The human hepatocellular carcinoma cells (HepG2) were treated with TiO2 NPs at doses of 0-200 μg/mL for 48 h and then RNA sequencing was implemented. The differential lncRNAs between the control and TiO2 NPs-treated groups were screened, then the lncRNA-mRNA network and enrichment pathways were analyzed via multivariate statistics. As a result, 46,759 lncRNAs were identified and 129 differential lncRNAs were screened out. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the targeted mRNAs of those differential lncRNAs were enriched in the Hedgehog signaling pathway, Vasopressin-regulated water reabsorption, and Glutamatergic synapse. Moreover, two lncRNA-mRNA networks, including lncRNA NONHSAT256380.1-JRK and lncRNA NONHSAT173563.1-SMIM22, were verified by mRNA detection. This study demonstrated that an alteration in the lncRNA expression profile could be induced by TiO2 NPs and epigenetics may play an important role in the mechanism of hepatotoxicity.

A cap 0-dependent mRNA capture method to analyze the yeast transcriptome.

Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes.

Identification and Functional Analysis of LncRNAs in Response to Seed Aging in Metasequoia glyptostroboides by Third Generation Sequencing Technology

The seeds of Metasequia glyptostroboides Hu et Cheng, an endangered species, are susceptible to aging, making natural population renewal difficult and increasing the risk of extinction. LncRNAs play important roles in plant growth and development and biotic and abiotic stress responses, but the functions of lncRNAs in the aging process of M. glyptostroboides seeds are still unclear. In this study, we used single molecule real-time (SMRT) sequencing technology in combination with Illumina RNA-seq to analyze lncRNA changes during M. glyptostroboides seed aging. We identified 403 intergenic lncRNAs (lincRNAs), 29 intronic lncRNAs, and 25 antisense lncRNAs; screened 9000 differentially expressed mRNAs (DEGs) and 128 differentially expressed lncRNAs (DELs); and predicted 844 cis-target genes and 8098 trans-target genes. GO and KEGG functional annotation of target genes revealed that the regulation of the reactive oxygen species metabolic process, protein processing in the endoplasmic reticulum, and the MAPK signaling pathway and other pathways were significantly enriched, showing a high correlation with the mRNA enrichment results. In addition, we constructed a ceRNA network consisting of 18 lncRNAs, 38 miRNAs, and 69 mRNAs, in which some miRNAs and mRNAs related to seed aging were found. Among them, miR167(a,b,c,d) may compete with lncRNA_00185, which is related to plant aging, to regulate the expression of the RCD1(Radical-induced Cell Death1) gene, thus promoting the balance of seed reactive oxygen species and enhancing seed-aging resistance. These results will have significant reference value in elucidating the molecular mechanism of the seed aging of M. glyptostroboides sequoia, improving the storage capacity for crop seeds, and protecting rare germplasm resources.

Region-specific changes in expression and activity of calpains in the CNS of native rats.

Introduction and Aim: It has been proposed that µ-calpain is responsible for neuronal survival, while m-calpain – for the degeneration. It can be assumed that the "susceptibility" to the damage factor for neurons in different CNS regions depends on the content/activity of calpain isoforms. We analyzed the mRNA levels and the activity of µ-and m-calpain in the different CNS structures of rats. Materials and Methods: After decapitation intact male Wistar rats the prefrontal cortex, striatum, hippocampus, midbrain, brainstem, cerebellum, and spinal cord were removed. Each structure was divided into two parts: casein zymography was performed to determine the activity and real-time RT–PCR - to determine the level of expression mRNA of µ-and m-calpains. Results: We have shown that m-calpain mRNA predominates in the striatum, midbrain and brainstem, while µ-calpain mRNA enrichment was noticed for the hippocampus and cerebellum. The highest µ-calpain activity was in the cervical spinal cord, the lowest - in the striatum. The m-calpain activity was relatively high in the midbrain, striatum, hippocampus and brainstem, while in the cervical spinal cord and cerebellum it was moderate. Conclusion: The selective neuronal death observed during neurodegeneration can be partially determined by the initial level of calpains expression and/or activity.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

Genome-Wide Analysis of the HDAC Gene Family and Its Functional Characterization at Low Temperatures in Tartary Buckwheat (Fagopyrum tataricum).

Histone deacetylases (HDACs), widely found in various types of eukaryotic cells, play crucial roles in biological process, including the biotic and abiotic stress responses in plants. However, no research on the HDACs of Fagopyrum tataricum has been reported. Here, 14 putative FtHDAC genes were identified and annotated in Fagopyrum tataricum. Their gene structure, motif composition, cis-acting elements, phylogenetic relationships, protein structure, alternative splicing events, subcellular localization and gene expression pattern were investigated. The gene structure showed FtHDACs were classified into three subfamilies. The promoter analysis revealed the presence of various cis-acting elements responsible for hormone, abiotic stress and developmental regulation for the specific induction of FtHDACs. Two duplication events were identified in FtHDA6-1, FtHDA6-2, and FtHDA19. The expression patterns of FtHDACs showed their correlation with the flavonoid synthesis pathway genes. In addition, alternative splicing, mRNA enrichment profiles and transgenic analysis showed the potential role of FtHDACs in cold responses. Our study characterized FtHDACs, providing a candidate gene family for agricultural breeding and crop improvement.

MiR-135a-5p suppresses trophoblast proliferative, migratory, invasive, and angiogenic activity in the context of unexplained spontaneous abortion

BackgroundSpontaneous abortions (SA) is amongst the most common complications associated with pregnancy in humans, and the underlying causes cannot be identified in roughly half of SA cases. We found miR-135a-5p to be significantly upregulated in SA-associated villus tissues, yet the function it plays in this context has yet to be clarified. This study explored the function of miR-135a-5p and its potential as a biomarker for unexplained SA.MethodRT-qPCR was employed for appraising miR-135a-5p expression within villus tissues with its clinical diagnostic values being assessed using ROC curves. The effects of miR-135a-5p in HTR-8/SVneo cells were analyzed via wound healing, Transwell, flow cytometry, EdU, CCK-8, and tube formation assays. Moreover, protein expression was examined via Western blotting, and interactions between miR-135a-5p and PTPN1 were explored through RIP-PCR, bioinformatics analyses and luciferase reporter assays.ResultsRelative to normal pregnancy (NP), villus tissue samples from pregnancies that ended in unexplained sporadic miscarriage (USM) or unexplained recurrent SA (URSA) exhibited miR-135a-5p upregulation. When this miRNA was overexpressed in HTR-8/SVneo cells, their migration, proliferation, and cell cycle progression were suppressed, as were their tube forming and invasive activities. miR-135a-5p over-expression also downregulated the protein level of cyclins, PTPN1, MMP2 and MMP9. In RIP-PCR assays, the Ago2 protein exhibited significant miR-135a-5p and PTPN1 mRNA enrichment, and dual-luciferase reporter assays indicated PTPN1 to be a bona fide miR-135a-5p target gene within HTR-8/SVneo cells.ConclusionmiR-135a-5p may suppress trophoblast migratory, invasive, proliferative, and angiogenic activity via targeting PTPN1, and it may thus offer value as a biomarker for unexplained SA.

Metatranscriptomics of cheese microbial communities: Efficiency of RNA extraction from various cheese types and of mRNA enrichment

Microbial communities from cheeses contribute to the development of typical organoleptic properties. Metatranscriptomic analyses can be used to provide a global picture of the functioning of these communities. Our objective was to evaluate the efficiency of RNA extraction from various cheese types and to evaluate mRNA enrichment procedures for metatranscriptomic analyses. For the 32 tested cheese brands, corresponding to five cheese types, the extraction yield varied from 1 μg to 363 μg RNA per gram of cheese and, overall, the yield was lower for fresh cheeses and for the core of pressed cooked cheeses than for the other cheese types. Pressed cooked cheeses also had a lower RNA integrity than the other cheese types. For total RNA extracts from four cheeses, approximately 99% of the sequencing reads corresponded to ribosomal RNA, and mRNA enrichment by RiboPOOL and FastSelect kits decreased this percentage to a range of 75 to 97% and of 53 to 76%, respectively. Comparison of RNA libraries after mRNA enrichment with libraries of undepleted total RNA showed that the FastSelect mRNA enrichment had a lower impact on the gene expression profiles of five target cheese species than the riboPOOL kit and the oligo (dT) selection method. The procedures that we describe in the present study may be useful for metatranscriptomic analysis of various cheese types.

Internal oligo(dT) priming introduces systematic bias in bulk and single-cell RNA sequencing count data.

Significant advances in RNA sequencing have been recently made possible by using oligo(dT) primers for simultaneous mRNA enrichment and reverse transcription priming. The associated increase in efficiency has enabled more economical bulk RNA sequencing methods and the advent of high-throughput single-cell RNA sequencing, already one of the most widely adopted methods in transcriptomics. However, the effects of off-target oligo(dT) priming on gene expression quantification have not been appreciated. In the present study, we describe the extent, the possible causes, and the consequences of internal oligo(dT) priming across multiple public datasets obtained from various bulk and single-cell RNA sequencing platforms. To explore and address this issue, we developed a computational algorithm for RNA counting methods, which identifies the sequencing read alignments that likely resulted from internal oligo(dT) priming and removes them from the data. Directly comparing filtered datasets to those obtained by an alternative method reveals significant improvements in gene expression measurement. Finally, we infer a list of human genes whose expression quantification is most likely to be affected by internal oligo(dT) priming and predict that when measured using these methods, the expression of most genes may be inflated by at least 10% whereby some genes are affected more than others.

Long noncoding RNA signatures involved in the genomic instability of papillary thyroid carcinoma

We aimed to identify long noncoding RNAs involved in the genomic instability of papillary thyroid carcinoma (PTC). Expression profiles of RNA-seq and gene mutation profiles were downloaded from the Cancer Genome Atlas (TCGA) database, and differentially expressed lncRNAs (DElncRNAs) and DE messenger RNAs (DEmRNAs) were determined. We constructed an lncRNA-mRNA network, analyzed mRNA enrichment, and compared the immune cell proportions and tumor mutation burdens between the low- and high-risk groups using a prognostic model. We identified 95 DElncRNAs and 421 DEmRNAs and constructed a network comprising 33 lncRNAs and 201 mRNAs. The mRNAs in the network were enriched in 36 Gene Ontology biological processes and 7 Kyoto Encyclopedia of Genes and Genomes pathways. A five-lncRNA prognostic model was constructed; the AUCs of the training, validation, and entire sets were 0.955, 0.805, and 0.901, respectively. The proportions of six types of tumor-infiltrating immune cells and neuroblastoma RAS viral (V-ras) oncogene homolog expression differed significantly between the low- and high-risk groups. LncRNAs may be involved in the genomic instability of PTC via cytokine-cytokine receptor interactions, cell adhesion molecules, and chemokine signaling pathways. Our five-lncRNA prognostic model may enable the prognostic evaluation of PTC patients. Highlights The 5-lncRNA prognostic model may be an independent prognostic factor for PTC. Six TIICs are associated with the prognosis of PTC. NRAS gene mutation plays a vital role in the progression of PTC. The model included the lncRNAs WARS2-IT1, LINC00536, ATP13A4-AS1, LINC01561, and FENDRR.

Differential mRNA Expression and Circular RNA-Based Competitive Endogenous RNA Networks in the Three Stages of Heart Failure in Transverse Aortic Constriction Mice.

BackgroundThe murine transverse aortic constriction (TAC) model is frequently used to investigate molecular mechanisms underlying heart failure. However, limited data is available regarding the expression of mRNAs and circRNAs in murine heart failure progression induced by pressure overload.MethodsTransverse aortic constriction was used to induce pressure overload for 2, 4, and 8 weeks in mice. Echocardiographic measurements in B-mode and M-mode, as well as blood flow Doppler data were collected in mice without (sham) and with (2W-, 4W-, and 8W-post-TAC) pressure load. Hearts were excised and morphology, cardiomyocyte size, and fibrosis were determined. RNA sequencing, circRNA microarray, functional mRNA enrichment analysis, hub gene identification, target miRNA interaction, and competitive endogenous RNA (ceRNA) network construction were conducted.ResultsHeart weight, cardiomyocyte hypertrophy, and fibrosis gradually increased over time in the hearts with pressure overload. The 2W-post-TAC hearts displayed concentric hypertrophy, thickened left ventricular walls, and increased EF and FS. The 4W-post-TAC hearts were characterized by preserved EF and FS, dilated atria, and increased left ventricle (LV) systolic volume. The 8W-post-TAC hearts presented with ventricular and atrial dilation, increased LV systolic and diastolic volume, reduced EF and FS, and increased ejection time (MV ET). mRNA expression analysis suggested that cardiac remodeling, immune response dysregulation, and metabolic disorder were the key cellular events in heart failure progression. Depression in chemotaxis and mitochondrial function were predicted in 4W- and 8W-post-TAC myocardia, respectively. A ceRNA network analysis demonstrated that the circRNAs targeted the expression of genes enriched in metabolism dysregulation in the 2W-post-TAC hypertrophic hearts, while they targeted genes enriched in cardiac remodeling in the 4W-post-TAC EF-preserved hearts and in the suppression of oxidative phosphorylation and cardiac contraction in the 8W-post-TAC EF-reduced hearts.ConclusionOur work empirically demonstrates that distinctive features of heart failure, including ventricular hypertrophy, heart failure with preserved EF (HFpEF), and heart failure with reduced EF (HFrEF) are present in the murine pressure overload models. The three stages of heart failure vary in terms of mRNA and circRNA expression, as well as ceRNA regulation in a manner consistent with their structural, functional, and pathological differences.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

AbstractAn athermal approach to mRNA enrichment from total RNA using a self‐immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six‐atom spacing between bases which allowed TENA to selectively base‐pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL−1. Self‐immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT‐qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT‐qPCR and other downstream molecular biology applications.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids.

An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL-1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.