Total RNA Samples Research Articles

Exhausted phenotype of follicular CD8 T cells in CVID

Exhausted phenotype of follicular CD8 T cells in CVID

DNA Framework-Mediated Electrochemical Biosensing Platform for Amplification-Free MicroRNA Analysis

MicroRNAs (miRNAs) have been explored as biomarkers for early diagnosis of diseases like cancers. However, it remains challenging to detect low-level miRNAs in the total RNA from real samples in a facile approach. In this work, we report a two-phase miRNA biosensing strategy based on a modular framework nucleic acid (FNA) platform, which combines the high efficiency of homogeneous reaction and the convenience of heterogeneous biosensing. In the first phase, free DNA probes bind target miRNAs in a homogeneous solution, forming a DNA-RNA complex with high base stacking energy. Then, at the second phase, the universal FNA interface on the electrode selectively mediated the transition of the complex from the solution onto the interface for electrochemical signal generating and transduction. By applying this method, we detected as few as 1 aM of miR-141, a cancer marker miRNA, without the need for nucleic acid amplification. The dynamic range spans 10 orders of magnitude. We demonstrate multiplex miRNA detection and discrimination of highly homologous miRNAs with mismatches as few as a single base. We also show that this system can detect miR-141 in only 50 ng of total RNA samples from real cells, which allows discrimination of prostate cancer cells with normal cells. We envision this platform may satisfy the need for facile and high-throughput screening of early cancer markers.

How to Perform miRacles: A Step-by-Step microRNA Detection Protocol Using DNA Nanoswitches.

MicroRNAs are short non-coding RNAs involved in post-transcriptional gene regulation, and are increasingly considered to be biomarkers for numerous biological processes and human diseases. Current techniques used for microRNA detection can be expensive and labor-intensive, and typically require amplification, labeling, or radioactive probes. In this protocol, we describe a DNA nanoswitch-based microRNA detection assay termed "miRacles": microRNA-activated conditional looping of engineered switches. This method uses conformationally responsive DNA nanoswitches that detect the presence of specific microRNAs with a simple and unambiguous gel-shift assay that can be performed on the benchtop. The assay is low cost, minimalistic, and capable of direct detection of specific microRNAs in unprocessed total RNA samples, with no enzymatic amplification, labeling, or special equipment. The protocol for detection of microRNAs in total RNA can be completed in as little as a few hours, making this assay a compelling alternative to qPCR and Northern blotting. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of DNA nanoswitches Basic Protocol 2: Detection of microRNAs from total RNA samples Support Protocol 1: Optional nanoswitch purification by PEG precipitation Support Protocol 2: Optional nanoswitch purification by liquid chromatography.

Effects of anterior thalamic nuclei stimulation on gene expression in a rat model of temporal lobe epilepsy.

Deep brain stimulation of the anterior nucleus of the thalamus (ANT-DBS) has been shown to be effective and safe in the long-term treatment of refractory epilepsy. However, the mechanisms by which ANT-DBS controls epilepsy at the gene expression level (e.g., which regulatory mechanisms are altered) is not well understood. Nine rats were randomly assigned to the control group, the kainic acid (KA) group, and the DBS group. Temporal lobe epilepsy in rats was induced by a stereotaxic KA injection (KA group). The DBS group received the KA injection followed by treatment with ANT-DBS. Video-electroencephalogram (EEG) was used to monitor seizures. Total RNA samples were isolated from the hippocampus of three groups. Microarray was used to detect differentially regulated mRNAs. GO and pathway analysis were performed to analyze the functional categories and affected pathways. qPCR was used to prove the reliability of the microarray results. The differentially expressed genes the KA group and the DBS group, relative to the control group, were screened and a total of 2910 genes were identified. These genes were involved in functional categories such as ion channel activity (P = 5.01 × 10-8), gated channel activity (P = 1.42 × 10-7), lipid binding (P = 4.97 × 10-5), and hydrolase activity (P = 5.02 × 10-5) and pathways such as calcium signaling pathway (P = 2.09 × 10-8), glutamatergic synapse (P = 4.09 × 10-8) and NOD-like receptor signaling pathway (P = 2.70 × 10-6). Differentially expressed mRNAs might play a role in the pathogenesis of temporal lobe epilepsy. Calcium signaling pathways, synaptic glutamate, and NOD-like receptor signaling pathway play a central role in normal-epilepsy-ANT-DBS treatment series. ANT-DBS achieves its antiepileptic effects by modulating target genes involved in a variety of functions and pathways.

Food authentication from shotgun sequencing reads with an application on high protein powders

Here we propose that using shotgun sequencing to examine food leads to accurate authentication of ingredients and detection of contaminants. To demonstrate this, we developed a bioinformatic pipeline, FASER (Food Authentication from SEquencing Reads), designed to resolve the relative composition of mixtures of eukaryotic species using RNA or DNA sequencing. Our comprehensive database includes >6000 plants and animals that may be present in food. FASER accurately identified eukaryotic species with 0.4% median absolute difference between observed and expected proportions on sequence data from various sources including sausage meat, plants, and fish. FASER was applied to 31 high protein powder raw factory ingredient total RNA samples. The samples mostly contained the expected source ingredient, chicken, while three samples unexpectedly contained pork and beef. Our results demonstrate that DNA/RNA sequencing of food ingredients, combined with a robust analysis, can be used to find contaminants and authenticate food ingredients in a single assay.

Maize transposable elements contribute to long non-coding RNAs that are regulatory hubs for abiotic stress response

BackgroundSeveral studies have mined short-read RNA sequencing datasets to identify long non-coding RNAs (lncRNAs), and others have focused on the function of individual lncRNAs in abiotic stress response. However, our understanding of the complement, function and origin of lncRNAs – and especially transposon derived lncRNAs (TE-lncRNAs) - in response to abiotic stress is still in its infancy.ResultsWe utilized a dataset of 127 RNA sequencing samples that included total RNA datasets and PacBio fl-cDNA data to discover lncRNAs in maize. Overall, we identified 23,309 candidate lncRNAs from polyA+ and total RNA samples, with a strong discovery bias within total RNA. The majority (65%) of the 23,309 lncRNAs had sequence similarity to transposable elements (TEs). Most had similarity to long-terminal-repeat retrotransposons from the Copia and Gypsy superfamilies, reflecting a high proportion of these elements in the genome. However, DNA transposons were enriched for lncRNAs relative to their genomic representation by ~ 2-fold. By assessing the fraction of lncRNAs that respond to abiotic stresses like heat, cold, salt and drought, we identified 1077 differentially expressed lncRNA transcripts, including 509 TE-lncRNAs. In general, the expression of these lncRNAs was significantly correlated with their nearest gene. By inferring co-expression networks across our large dataset, we found that 39 lncRNAs are as major hubs in co-expression networks that respond to abiotic stress, and 18 appear to be derived from TEs.ConclusionsOur results show that lncRNAs are enriched in total RNA samples, that most (65%) are derived from TEs, that at least 1077 are differentially expressed during abiotic stress, and that 39 are hubs in co-expression networks, including a small number that are evolutionary conserved. These results suggest that lncRNAs, including TE-lncRNAs, may play key regulatory roles in moderating abiotic responses.

Genetic profiling of Hsp70 gene in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India.

This study was aimed to genetic profiling of heat shock protein 70 (Hsp70) gene in Murrah buffalo investigating 50 unrelated adult animals at ICAR-Research Complex for Eastern Region, Patna (India) in winter, spring, and summer. PCR ready genomic DNA samples and season-wise total RNA samples were prepared. The PCR products of Hsp70 eluted from agarose gel were sequenced and analyzed. The first-strand cDNA was synthesized and concentration was equalized to 25 ng/μl. Expression kinetics of mRNA transcripts in different seasons was studied using Brilliant SYBR Green QPCR technique and the data retrieved was analyzed by least-squares ANOVA. DNA sequencing by primer walking revealed four allelic variants of Hsp70 gene. Alignment study revealed one substitution in 5'UTR, six substitutions in coding region, and one addition in 3'UTR. The highest percent identity and negligible phylogenetic distance were found among the alleles and reference bovine sequences. The relative mRNA expression was significantly higher in summer when THI ≥ 84 than the spring and winter; fold change increased by 4.5 times in summer than the spring whereas found nearly half in winter. These findings can be useful for heat stress management in buffaloes and help in understanding the mechanism of thermo-regulation well.

Droplet microfluidic platform for fast and continuous-flow RT-qPCR analysis devoted to cancer diagnosis application

RT-qPCR represents a key method in cancer diagnostic, however the constant increase in patients and cancer biomarker panels to screen requires the implementation of faster approaches allowing smaller reagents and samples volumes consumption. To fulfil these needs, we present here a fully automated droplet microfluidics platform that couples a specifically designed thermal system with a fluorescence excitation/detection module. Additionally, the droplet generation and merging approaches allows the analysis of multiple samples and genes with no risk of contamination. This platform has been initially validated by investigating HER2 overexpression in two cancer cell lines starting from total RNA samples; these results have been successfully compared with those obtained by a commercially available machine, showing no limitation in terms of number of processed samples as well as reducing of 200 times the analysis costs per patient. Finally, we have demonstrated its capability in performing fast RT-qPCR, raising the throughput of analysis to a hundred samples in less than 20 min.

Systematic assessment of commercially available low-input miRNA library preparation kits

ABSTRACTHigh-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

Ionic Liquid Modified Single‐use Electrode Developed for Voltammetric Detection of miRNA‐34a and its Application to Real Samples

AbstractThe ionic liquid (IL) modified chemically activated (CA) pencil graphite electrodes (PGEs) were developed for label‐free voltammetric detection of miRNA‐34a, and implemented to the real samples. Firstly, the electrochemical characterization of unmodified PGE, CA‐PGE, IL‐PGE and IL‐CA‐PGE was performed by cyclic voltammetry (CV) as well as their DNA binding capacity was studied by electrochemical impedance spectroscopy (EIS) technique. The microscopic characterization of the surface of each electrodes was investigated by scanning electron microscopy (SEM). Differential pulse voltammetry (DPV) technique was used for measuring the oxidation signal of guanine in order to perform a label‐free voltammetric monitoring of a full‐match hybridization specific to miRNA‐34a. The selectivity of biosensor was tested against to miRNA‐155, miRNA‐660 as well as to the mismatch sequence of miRNA‐34a. The further selectivity of this proposed biosensor was studied in the mixture of samples containing miRNA‐34a with other miRNAs (1 : 1). The voltammetric detection of miRNA‐34a was also explored in the artificial serum medium as fetal bovine serum (FBS) and also in total RNA samples isolated from HUH‐7 human hepatocellular carcinoma cell line.

Signal-on electrogenerated chemiluminescence biosensor for ultrasensitive detection of microRNA-21 based on isothermal strand-displacement polymerase reaction and bridge DNA-gold nanoparticles

MicroRNA-21 (miRNA-21) is a promising diagnostic biomarker for breast cancer screening and disease progression. A sensitive and selective strategy for the quantitative determination of miRNA-21 is of great significance in the early diagnosis of cancers. Herein, a novel electrogenerated chemiluminescence (ECL) biosensor was designed for the detection of miRNA-21 with dual signal amplification based on isothermal strand-displacement polymerase reaction (ISDPR) and bridge DNA-gold nanoparticles (AuNPs) nanocomposites. The ECL biosensor was fabricated by self-assembling a thiolated capture probe (SH-CP) on the surface of a gold electrode. The target miRNA-21 initiated the phi29 DNA polymerase-mediated ISDPR, which could generate large numbers of single-stranded DNA (assistant DNA) with accurate and comprehensive nucleotide sequences. The assistant DNA was captured by the SH-CP self-assembled on the Au electrode and further hybridized with bridge DNA-AuNPs nanocomposites, more biotins can be captured on the electrode surface. Afterward, a streptavidin-modified Ru (bpy)32+ complex (SA-Ru) was bound to the bridge DNA-AuNPs nanocomposites via a specific interaction between biotin and streptavidin to produce a strong ECL signal. The ECL intensity was logarithmically proportion to the concentration of target miRNA-21 over a range from 0.01 fM to 10,000 fM with a detection limit of 3.2 aM. The proposed ECL biosensor was successfully applied to detect miRNA-21 in total RNA samples extracted from human breast cancer cells, and it showed great potential for early cancer diagnosis based on miRNA as a biomarker.

MYC-driven regulation of long non-coding RNA profiles in breast cancer cells

AimMYC deregulation contributes to breast cancer development and progression. Deregulated expression levels of long non-coding RNAs (lncRNA) have been demonstrated to be critical players in development and/or maintenance of breast cancer. In this study we aimed to evaluate lncRNA expressions depending on MYC overexpression and knockdown in breast cancer cells. Materials and methodsCells were infected with lentiviral vectors by either knockdown or overexpression of c-MYC. LncRNA cDNA was transcribed from total RNA samples and lncRNAs were evaluated by qRT-PCR. ResultsOur results indicated that some of the lncRNAs having tumor suppressor (GAS5, MEG3, lincRNA-p21) and oncogenic roles (HOTAIR) are regulated by c-MYC. ConclusionWe observed that c-MYC regulates lncRNAs that have important roles on proliferation, cell cycle and etc. Further studies will give us a light to identify molecular mechanisms related to MYC-lncRNA regulatory pathways in breast cancer.

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Small intestinal transcriptome analysis revealed changes of genes involved in nutrition metabolism and immune responses in growth retardation piglets1

Postnatal growth retardation (PGR) is common in piglets. Abnormal development in small intestine was casually implicated in impaired growth, but the exact mechanism is still implausible. The present study unveiled transcriptome profile of jejunal mucosa, the major site of nutrient absorption, in PGR and healthy piglets using RNA-sequencing (RNA-seq). The middle segments of jejunum and ileum, and jejunal mucosa were obtained from healthy and PGR piglets at 42 d of age. Total RNA samples extracted from jejunal mucosa of healthy and PGR piglets were submitted for RNA-seq. Lower villus height was observed in both jejunum and ileum from PGR piglets suggesting structural impairment in small intestine (P < 0.05). RNA-seq libraries were constructed and sequenced, and produced average 4.8 × 107 clean reads. Analysis revealed a total of 499 differently expressed genes (DEGs), of which 320 DEGs were downregulated in PGR piglets as compared to healthy piglets. The functional annotation based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) highlighted that most DEGs were involved in nutrient metabolism and immune responses. Our results further indicated decreased gene expression associated with glucose, lipid, protein, mineral, and vitamin metabolic process, detoxication ability, oxidoreductase activity, and mucosal barrier function; as well as the increased insulin resistance and inflammatory response in the jejunal mucosa of PGR piglets. These results characterized the transcriptomic profile of the jejunal mucosa in PGR piglets, and could provide valuable information with respect to better understanding the nutrition metabolism and immune responses in the small intestine of piglets.

ArcasHLA: high-resolution HLA typing from RNAseq.

MotivationThe human leukocyte antigen (HLA) locus plays a critical role in tissue compatibility and regulates the host response to many diseases, including cancers and autoimmune di3orders. Recent improvements in the quality and accessibility of next-generation sequencing have made HLA typing from standard short-read data practical. However, this task remains challenging given the high level of polymorphism and homology between HLA genes. HLA typing from RNA sequencing is further complicated by post-transcriptional modifications and bias due to amplification.ResultsHere, we present arcasHLA: a fast and accurate in silico tool that infers HLA genotypes from RNA-sequencing data. Our tool outperforms established tools on the gold-standard benchmark dataset for HLA typing in terms of both accuracy and speed, with an accuracy rate of 100% at two-field resolution for Class I genes, and over 99.7% for Class II. Furthermore, we evaluate the performance of our tool on a new biological dataset of 447 single-end total RNA samples from nasopharyngeal swabs, and establish the applicability of arcasHLA in metatranscriptome studies.Availability and implementationarcasHLA is available at https://github.com/RabadanLab/arcasHLA.Supplementary information Supplementary data are available at Bioinformatics online.

Similarities in mRNA expression of peripheral blood drug metabolizing enzymes and cancer marker genes with biopsy samples of head and neck cancer patients

Purpose: To develop peripheral blood mRNA expression profiles of drug metabolizing enzymes (DMEs) as a surrogate to monitor tobacco induced head and neck squamous cell carcinoma (HNSCC), attempts were made to investigate (i) similarities in alterations with the cancer marker genes in biopsy samples and (ii) if alterations similar to that seen in biopsy samples are reflected in peripheral blood.Methods: Total RNA from eight soft gingival tissues and eight biopsy samples of HNSCC patients and total DNA and RNA from blood of healthy controls (n = 150) and HNSCC patients (n = 150) was processed for expression and genotyping studies. Blood from patients receiving chemo-radiotherapy was processed for follow-up study.Results: qRT-PCR revealed significant increase in mRNA expression of DMEs in biopsy and blood samples of HNSCC patients when compared to controls. Similar alterations were observed in cancer marker genes in these samples. Patients with variant genotypes of DMEs showed greater magnitude of alterations in mRNA expression when compared to wild type controls. Responders of chemo-radiotherapy showed significant decline in induction of mRNA expression of DMEs and cancer marker genesConclusions: The data suggest that peripheral blood expression profiles could be used to monitor tobacco-induced HNSCC as well as the treatment response.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

A novel species of RNA virus associated with root lesion nematode Pratylenchus penetrans.

The root lesion nematode Pratylenchus penetrans is a migratory species that attacks a broad range of plants. While analysing transcriptomic datasets of P. penetrans, we have identified a full-length genome of an unknown positive-sense single-stranded RNA virus, provisionally named root lesion nematode virus 1 (RLNV1). The 8614-nucleotide genome sequence encodes a single large polyprotein with conserved domains characteristic for the families Picornaviridae, Iflaviridae and Secoviridae of the order Picornavirales. Phylogenetic, BLAST and domain search analyses showed that RLNV1 is a novel species, most closely related to the recently identified sugar beet cyst nematode virus 1 and potato cyst nematode picorna-like virus. In situ hybridization with a DIG-labelled DNA probe confirmed the presence of the virus within the nematodes. A negative-strand-specific RT-PCR assay detected RLNV1 RNA in nematode total RNA samples, thus indicating that viral replication occurs in P. penetrans. To the best of our knowledge, RLNV1 is the first virus identified in Pratylenchus spp.

Monoallelic, antisense and total RNA transcription in an in vitro neural differentiation system based on F1 hybrid mice.

We developed an in vitro system to differentiate embryonic stem cells (ESCs) derived from reciprocally crossed F1 hybrid mice into neurons, and used it to investigate poly(A)+ and total RNA transcription at different stages of cell differentiation. By comparing expression profiles of transcripts assembled from 20 RNA sequencing datasets [2 alleles×(2 cell lines×4 time-points+2 mouse brains)], the relative influence of strain, cell and parent specificities to overall expression could be assessed. Divergent expression profiles of ESCs converged tightly at neural progenitor stage. Patterns of temporal variation of monoallelically expressed transcripts and antisense transcripts were quantified. Comparison of sense and antisense transcript pairs within the poly(A)+ sample, within the total RNA sample, and across poly(A)+ and total RNA samples revealed distinct rates of pairs showing anti-correlated expression variation. Unique patterns of sharing of poly(A)+ and poly(A)- transcription were identified in distinct RNA species. Regulation and functionality of monoallelic expression, antisense transcripts and poly(A)- transcription remain elusive. We demonstrated the effectiveness of our approach to capture these transcriptional activities, and provided new resources to elucidate the mammalian developmental transcriptome.

Gene expression profiles in the dorsal root ganglia of methylmercury-exposed rats.

Methylmercury (MeHg) exposure is known to induce neurodegeneration in both the central nervous system (CNS) and peripheral nervous system (PNS). Molecular mechanisms of MeHg-induced neurotoxicity have been well investigated in the CNS, however, it remains unclear in the PNS. In the present study, comprehensive gene expression analysis was performed by analyzing MeHg-exposed adult rat dorsal root ganglion (DRG) by DNA microarray. Methylmercuric chloride (6.7 mg/kg/day) was administered to nine-week-old male Wistar rats for five days, followed by two days without administration; this cycle was repeated once. Rats were anesthetized at 7 or 14 days after commencement of MeHg exposure, and their DRGs were removed and homogenized to make total RNA samples. DNA microarray data from Day 7 samples identified 100 out of 18,513 detected genes as annotated genes with more than two-fold upregulated or downregulated expression compared with controls. Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested strong involvement of immune activation and inflammation pathways in rat DRG exposed to MeHg, and some genes overlapped with previously reported genes affected by MeHg exposure in the cerebellum. The present results suggest that MeHg-induced neurotoxicity is associated with immune activation and inflammatory responses in rat DRG.