A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations.
Ontology highlight
ABSTRACT:
SUBMITTER: Mackie C
PROVIDER: S-EPMC1186465 | biostudies-other | 1979 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:We have adapted the CyQuant(R) assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant(R) assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (>1000) of adherent cells.
| S-EPMC2721084 | biostudies-other
Project description:A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies.
| S-EPMC3695942 | biostudies-literature
Project description:The RNA of the blue-green alga Anacystis nidulans contains three ribosomal RNA species with molecular weights of 0.56x10(6), 0.9x10(6), and 1.1x10(6) if the RNA is extracted in the absence of Mg(2+). The 0.9x10(6)mol.wt. rRNA is extremely slowly labelled in (32)P-incorporation experiments. This rRNA may be a cleavage product of the 1.1x10(6)mol.wt. rRNA from the ribosomes of cells in certain physiological states (e.g. light-deficiency during growth). The cleavage of the 1.1x10(6)mol.wt. rRNA during the extraction procedure can be prevented by the addition of 10mm-MgCl(2). (32)P-pulse-labelling studies demonstrate the rapid synthesis of two ribosomal precursor RNA species. One precursor RNA migrating slightly slower than the 1.1x10(6)mol.wt. rRNA appears much less stable than the other precursor RNA, which shows the electrophoretic behaviour of the 0.7x10(6)mol.wt. rRNA. Our observations support the close relationship between bacteria and blue-green algae also with respect to rRNA maturation. The conversion of the ribosomal precursor RNA species into 0.56x10(6)- and 1.1x10(6)-mol.wt. rRNA species requires Mg(2+) in the incubation medium.
| S-EPMC1174049 | biostudies-other
Project description:An improved one-step method for the extraction of RNA from rat brain is described. Fresh or frozen tissue is disrupted in the powerful protein denaturant guanidine thiocyanate, and RNA isolated by ultracentrifugation through CsCl. The procedure is advantageous in that it is relatively simple, is rapid and does not expose the sample to enzyme treatments or repeated organic extractions.
| S-EPMC1161490 | biostudies-other
Project description:A method is described for measuring the porportion of a specific messenger RNA in the total RNA extracted from pulse-labelled cells. A model system consisting of total ribosomal RNA and Escherichia coli DNA is used to validate the method and to define the conditions under which it can be used.
| S-EPMC1176491 | biostudies-other
Project description:PurposeTo clarify the short-term effect of topical anesthetics on 16S ribosomal ribonucleic acid amplicon sequencing results in ocular surface microbiome research.MethodsBoth eyes of 24 eligible volunteers undergoing general anesthesia were sampled. Before sampling, a drop of artificial tears or a drop of topical anesthetic was applied in a randomized way. By using artificial tears as a control, we assured blinding of the executer and took a potential diluting effect into account. Bacterial DNA was extracted using the QIAGEN RNeasy PowerMicrobiome Kit with specific adaptations. Amplified DNA was sequenced with the Illumina MiSeq sequencing platform.ResultsFour sample pairs were excluded due to low yield of bacterial DNA. In the remaining 20 sample pairs, no differences were observed with topical anesthetics at the levels of amplicon sequence variants (ASVs), phylum, genera, or alpha and beta diversity. Weighted UniFrac distance confirmed that the intraindividual distance between the right and left eye was smaller than the effect of the topical anesthetic. Interestingly, however, we identified Cutibacterium as a potential discriminative biomarker for topical anesthetic use. Overall, a significantly higher number of observed reads were assigned to genera with Gram-positive characteristics.ConclusionsBased on our targeted, double-blinded, within-subject study, topical anesthetics do not affect the overall sequencing results but display a specific effect on Cutibacterium. When comparing research results, the impact of topical anesthetics on prevalence and abundance of Cutibacterium should be considered.Translational relevanceUnderstanding and standardization of sampling techniques are indispensable to properly execute clinical microbiome research.
| S-EPMC8899854 | biostudies-literature
Project description:Liquid-Liquid Phase Separation (LLPS) plays a crucial role in cell biology and is closely associated with neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Recent studies connect mutations in the C9ORF72 gene to the production of arginine-rich dipeptide repeat proteins (R-DPRs), such as poly(PR) and poly(GR). These R-DPRs disrupt LLPS in membrane-less organelles (MLOs) and contribute to disease pathology. While traditional analysis techniques like nuclear magnetic resonance (NMR), fluorescence recovery after photobleaching (FRAP), and Förster resonance energy transfer (FRET) provide insights into LLPS's role in these diseases, their ability is limited in detecting weak intermolecular interactions within LLPS droplets. This study employs graphene field-effect transistors (GFETs) for their superior sensitivity in detecting these molecular interactions. We immobilized RNA (poly-A) on GFETs and measured the electrical conductivity of GFETs to characterize shifts in the voltage of the charge neutral point in GFETs, allowing for the detection of dipeptide repeat peptides, such as (PR)12, (GR)12, and R12. Our results show that interactions between peptides and RNA require a specific peptide concentration threshold and vary between peptide types. Notably, the minimal conductivity shift suggests that peptides containing proline residues exhibit a nonuniform spatial distribution during interactions with RNA on graphene surfaces. This finding indicates that peptide rigidity induced by prolines plays a vital role in these molecular interactions and their multivalent contacts with RNA, which agrees with findings reported in other recent works. The capability of GFETs to detect these interactions at nanomolar concentrations marks a significant advancement in sensitivity over existing methods. This research sheds light on the mechanisms of LLPS involving R-DPRs and opens avenues for further understanding of related neurodegenerative diseases.
| S-EPMC11874026 | biostudies-literature
Project description:Noncoding RNA (ncRNA) is a kind of RNA that plays an important role in many biological processes, diseases, and cancers, while cannot translate into proteins. With the development of next-generation sequence technology, thousands of novel RNAs with long open reading frames (ORFs, longest ORF length > 303 nt) and short ORFs (longest ORF length ≤ 303 nt) have been discovered in a short time. How to identify ncRNAs more precisely from novel unannotated RNAs is an important step for RNA functional analysis, RNA regulation, etc. However, most previous methods only utilize the information of sequence features. Meanwhile, most of them have focused on long-ORF RNA sequences, but not adapted to short-ORF RNA sequences. In this paper, we propose a new reliable method called NCResNet. NCResNet employs 57 hybrid features of four categories as inputs, including sequence, protein, RNA structure, and RNA physicochemical properties, and introduces feature enhancement and deep feature learning policies in a neural net model to adapt to this problem. The experiments on benchmark datasets of 8 species shows NCResNet has higher accuracy and higher Matthews correlation coefficient (MCC) compared with other state-of-the-art methods. Particularly, on four short-ORF RNA sequence datasets, specifically mouse, Saccharomyces cerevisiae, zebrafish, and cow, NCResNet achieves greater than 10 and 15% improvements over other state-of-the-art methods in terms of accuracy and MCC. Meanwhile, for long-ORF RNA sequence datasets, NCResNet also has better accuracy and MCC than other state-of-the-art methods on most test datasets. Codes and data are available at https://github.com/abcair/NCResNet.
| S-EPMC7059790 | biostudies-literature
Project description:Background and aimSupplementation of AKBISprob (developed in a previous study) in feed can improve production efficiency and poultry health, especially laying hens. In addition, it can also increase cellulolytic lactic acid bacteria (LAB) in chicken intestines, but these bacteria are still unknown; thus, they need to be identified. This study aimed to identify cellulolytic LAB in the intestines of laying hens administered AKBISprob based on 16S ribosomal ribonucleic acid (16S rRNA) gene analysis.Materials and methodsThe samples used in this study were 13 LAB isolates from the intestines of laying hens that were given AKBISprob 4%. Cellulolytic LAB DNA was isolated and 16S rRNA gene was amplified by polymerase chain reaction, followed by sequencing, bioinformatics analysis, and phylogenetic tree construction.ResultsFrom 10 cellulolytic LAB isolates with a clear zone of >6 mm, four were selected and their DNA was amplified with BaCF and UniB primers ~1500 bp DNA fragments. Of these, the P31H62 isolate was genetically close to Enterococcus hirae strain 1-1X-16 with 92.90% maximum identity, the P33S52 isolate had homology with Enterococcus mundtii strain ZU 26 with 96.76% maximum identity, and the P33S62 isolate was closely related to E. hirae strain SJ3 with 72.96% maximum identity. The phylogenetic tree revealed that the cellulolytic LAB isolates P31H62 and P33S52 were in one cluster closely related to the genus Enterococcus.ConclusionThis study suggests that the isolates P31H62, P33S62, and P33S52 from the intestines of laying hens administered 4% AKBISprob are cellulolytic LAB belonging to the genus Enterococcus.
| S-EPMC9394149 | biostudies-literature
Project description:Tracking active transcription with the nuclear run-on (NRO) assays has been instrumental in uncovering mechanisms of gene regulation. The coupling of NROs with high-throughput sequencing has facilitated the discovery of previously unannotated or undetectable RNA classes genome-wide. Precision run-on sequencing (PRO-seq) is a run-on variant that maps polymerase active sites with nucleotide or near-nucleotide resolution. One main drawback to this and many other nascent RNA detection methods is the somewhat intimidating multi-day workflow associated with creating the libraries suitable for high-throughput sequencing. Here, we present an improved PRO-seq protocol where many of the enzymatic steps are carried out while the NRO RNA remains bound to magnetic beads. These adaptations reduce time, and we demonstrate that the resulting libraries are of the same quality as libraries generated using the original published protocol. The faster library preparation procedure decreases opportunity for sample loss and RNA degradation.
2020-05-20 | GSE150625 | GEO