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At least 469 records · Page 26Linked to original sources
PubMed1984

Inhibition of ribonuclease contamination in preparations of T4 RNA ligase, polynucleotide kinase, and bacterial alkaline phosphatase with bentonite.

Commercial preparations of the enzymes used in the analysis of RNA primary structure (bacterial alkaline phosphatase, polynucleotide kinase, and RNA ligase) are virtually always more or less contaminated with RNases. This leads to degradation of initial RNAs in the course of labeling and formation of a set of spurious labeled fragments. We have shown that bentonite present in the incubation medium in a concentration of 0.04% selectively inhibits the contaminating RNases, not affecting the activities of bacterial alkaline phosphatase, polynucleotide kinase, and RNA ligase.

Alkaline Phosphatase↗
PubMed1997

RnaViz, a program for the visualisation of RNA secondary structure.

RnaViz is a user-friendly, portable, windows-type program for producing publication-quality secondary structure drawings of RNA molecules. Drawings can be created starting from DCSE alignment files if they incorporate structure information or from mfold ct files. The layout of a structure can be changed easily. Display of special structural elements such as pseudo-knots or unformatted areas is possible. Sequences can be automatically numbered, and several other types of labels can be used to annotate particular bases or areas. Although the program does not try to produce an initially non-overlapping drawing, the layout of a properly positioned structure drawing can be applied to a newly created drawing using skeleton files. In this way a range of similar structures can be drawn with a minimum of effort. Skeletons for several types of RNA molecule are included with the program.

Base Sequence↗
PubMed2001

Quantitation of viral load using real-time amplification techniques.

Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA)-based systems combined with several detection strategies are being employed in a clinical diagnostic setting. The importance of these assays in disease management is still in an exploration phase. Although these technologies have the implicit capability of accurately measuring DNA and RNA in clinical samples, issues related to standardization and quality control must be resolved to enable routine implementation of these technologies in molecular diagnostics.

Calibration↗
PubMed1988

A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis.

A small-scale method for the isolation of total RNA from plant tissue is described. The method provides RNA of suitable quantity and quality from 0.2 g fresh tissue for the detection of mRNA species by RNA blot analysis. The entire procedure is adapted to 1.5-ml microfuge tubes and takes less than 5 h. This method is well suited for the isolation of RNA from large numbers of samples or from samples of limited quantity.

Blotting, Northern↗
PubMed2006

Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proficiency panel.

In recent years testing responsibilities for high-consequence pathogens have been expanded from national reference laboratories into networks of local and regional laboratories in order to support enhanced disease surveillance and to test for surge capacity. This movement of testing of select agents and high-consequence pathogens beyond reference laboratories introduces a critical need for standardized, noninfectious surrogates of disease agents for use as training and proficiency test samples. In this study, reverse transcription-PCR assay RNA targets were developed and packaged as armored RNA for use as a noninfectious, quantifiable synthetic substitute for four high-consequence animal pathogens: classical swine fever virus; foot-and-mouth disease virus; vesicular stomatitis virus, New Jersey serogroup; and vesicular stomatitis virus, Indiana serogroup. Armored RNA spiked into oral swab fluid specimens mimicked virus-positive clinical material through all stages of the reverse transcription-PCR testing process, including RNA recovery by four different commercial extraction procedures, reverse transcription, PCR amplification, and real-time detection at target concentrations consistent with the dynamic ranges of the existing real-time PCR assays. The armored RNA concentrations spiked into the oral swab fluid specimens were stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling of proficiency panel samples, including 24 h at 37 degrees C and 2 weeks at temperatures ranging from ambient room temperature to -70 degrees C. The analytic test performance, including the reproducibility over the dynamic range of the assays, indicates that armored RNA can provide a noninfectious, quantifiable, and stable virus surrogate for specific assay training and proficiency test purposes.

Laboratories↗
PubMed2002

Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA.

Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription#150;PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 x 10(11)-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.

Cluster Analysis↗
PubMed1993

Direct extraction and purification of rRNA for ecological studies.

Microgram quantities of rRNA were recovered from natural microbial communities in sediment, soil, and water with a lysozyme-hot phenol direct extraction method. Gel filtration with Sephadex G-75 spun columns readily removed humic-like contaminants without any measurable loss of rRNA and rendered RNA extracts of sufficient quality for molecular procedures.

Bacteria↗
PubMed2025

Assessment of genomic prediction capabilities of transcriptome data in a barley multi-parent RIL population.

Low-cost and high-throughput RNA sequencing data for barley RILs achieved GP performance comparable to or better than traditional SNP array datasets when combined with parental whole-genome sequencing SNP data. The field of genomic selection (GS) is advancing rapidly on many fronts including the utilization of multi-omics datasets with the goal of increasing prediction ability and becoming an integral part of an increasing number of breeding programs ensuring future food security. In this study, we used RNA sequencing (RNA-Seq) data to perform genomic prediction (GP) on three related barley RIL populations. We investigated the potential of increasing prediction ability by combining genomic and transcriptomic datasets, adding whole-genome sequencing (WGS) SNP data, functional annotation-based filtering, and empirical quality filtering. Our RNA-Seq data were generated cost-efficiently using small-footprint plant cultivation, high-throughput RNA extraction, and Library preparation miniaturization. We also examined sequencing depth reduction as an additional cost-saving measure. We used fivefold cross-validation to evaluate the prediction ability of the gene expression dataset, the RNA-Seq SNP dataset, and the consensus SNP dataset between the RNA-Seq and parental WGS data, resulting in prediction abilities between 0.73 and 0.78. The consensus SNP dataset performed best, with five out of eight traits performing significantly better compared to a 50K SNP array, which served as a benchmark. The advantage of the consensus SNP dataset was most prominent in the inter-population predictions, in which the training and validation sets originated from different RIL sub-populations. We were therefore able to not only show that RNA-Seq data alone are able to predict various complex traits in barley using RILs, but also that the performance can be further increased with WGS data for which the public availability will steadily increase.

Hordeum↗
PubMed2000

Biological stability of RNA isolated from RNAlater-treated brain tumor and neuroblastoma xenografts.

BACKGROUND: Advances in molecular biological research have led to identification of prognostic factors such as Trk mRNA expression in primitive neuroectodermal tumors of the CNS and neuroblastoma. To study prospectively the importance of these prognostic factors in large groups of homogeneously treated patients, tumor specimens of good quality must be acquired, preserved, and stored at multiple institutions. Immediate freezing of tumor biopsy samples in liquid nitrogen and storage at -70 degrees C are the most commonly used method of tissue preservation for future RNA analysis. PROCEDURE: To evaluate alternative methods of preserving tissue samples for subsequent RNA analysis, we tested a new RNA stabilization solution. Using tumor tissue of two CNS tumor and one neuroblastoma human xenografts, we compared total RNA isolated from tumor tissue stored for 7 days at room temperature in stabilization solution to that of snap-frozen tissue. The quality of the RNA was studied by spectrophotometry, gel electrophoresis, RT-PCR, and gene expression profiling. RESULTS: No major differences were observed in the quality of RNA isolated from tumor samples stored at room temperature in the RNA stabilization solution compared to snap-frozen tumor samples stored at -70 degrees C. CONCLUSIONS: High-quality RNA can be prepared from tumor tissue stored at room temperature. Whenever snap freezing is not feasible, pieces of tumor tissue can be treated with RNAlater for subsequent RNA analysis. Short-term storage and shipment of well-preserved tumor tissue are clearly feasible for all institutions, thereby facilitating large multiinstitutional studies of biological prognostic factors.

Animals↗
PubMed2000

Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA.

Reverse transcription polymerase chain reaction (RT-PCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol, RNAzol, FastTube reagent). RNA yield was slightly higher with RNAzol than with TRIzol as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol and TRIzol. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol, and 1 BCR-ABL-positive (specific for translocation t [9; 221) cell among 2x10(4) normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.

Base Sequence↗
PubMed2025

Dual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host-bacterial gene expression during sepsis. The DRIB protocol yielded 2.10-6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6-24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51-68% (8.4-10.9 million) reads, while 0.5-6.7% (79,496-789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host-bacterial gene co-expression dynamics in sepsis progression and outcomes.

Humans↗
PubMed1999

Standardization: a progress report.

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.

Blood Banks↗
PubMedDate not supplied

Molecular methods for identification and detection of bacterial food pathogens.

The polymerase chain reaction (PCR) shortens conventional microbiological methods for the detection of food pathogens either by replacing the conventional biochemical and serological identification or by its direct use on pre-enrichment media or food products. PCR allows fast and highly reliable identification of bacterial taxa, particularly phenotypically atypical bacterial strains. For reliablity, PCR primers and reaction conditions must be thoroughly optimized and evaluated, appropriate sample preparations must be developed, and a stringent laboratory protocol must be followed. Positive control systems are used to monitor possible inhibition of the reaction and negative controls are needed to monitor for contamination. The most recent developments involve messenger RNA-based (mRNA-based) detection of viable bacterial pathogens and real-time PCR quantitation of pathogens.

Bacteria↗
PubMed2003

Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities.

Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

Arachis↗
PubMed2004

The effect of oocyte quality on development.

Oocyte quality affects early embryonic survival, the establishment and maintenance of pregnancy, fetal development, and even adult disease. Quality, or developmental competence, is acquired during folliculogenesis as the oocyte grows, and during the period of oocyte maturation. Assisted reproductive technologies involving ovarian hyperstimulation, or collection of immature oocytes followed by maturation in vitro, perturb this process and result in oocytes with reduced quality. In domestic livestock species, offspring have been produced using in vitro oocyte maturation, although only a small percentage of the original pool of immature oocytes is capable of developing to the blastocyst stage and subsequently resulting in pregnancy. In vitro maturation, as it is currently undertaken, does not support the correct development of oocyte competence. Follicle size affects oocyte quality, potentially implicating messenger RNA or protein stores as factors involved in oocyte competence. Oocytes from preantral follicles grown in vitro are competent to resume meiosis, although development to the blastocyst stage is decreased. An offspring from oocytes produced using this technique was normal at birth but experienced delayed onset health issues, highlighting the importance of oocyte quality long after embryogenesis. Metabolism may play a critical role in oocyte quality because glycolytic activity in mature oocytes is correlated with increased embryonic development. Communication between the oocyte and its surrounding cumulus cells is also important for the development of a competent oocyte. Ovarian stimulation causes delayed embryonic development, increased abnormal blastocyst formation, fetal growth retardation, and increased fetal loss. Thus, although meiosis and even early development may be completed successfully, there are a variety of other processes occurring within the cytoplasm of the oocyte that are required for complete developmental competence. However, the cellular mechanisms that impart oocyte quality are unclear. Until the mechanisms involved in oocyte quality are elucidated, any effort to use assisted reproductive technologies in animals for production or biomedical purposes will be inefficient at best.

Animals↗
PubMed2003

Precipitation of RNA impurities with high salt in a plasmid DNA purification process: use of experimental design to determine reaction conditions.

The use of high salt solution to precipitate RNA in a pharmaceutical-grade plasmid DNA purification process was investigated. Five antichaotropic salts were tested for their potential to precipitate RNA. Calcium chloride was by far the best precipitant with high RNA removal in a very short incubation time. Calcium chloride precipitation conditions were investigated at two stages of a plasmid purification process using experimental design techniques. The effect of up to five factors on RNA precipitation and plasmid recovery was assessed by statistical modeling. Optimized conditions for calcium chloride precipitation were then introduced to the plasmid purification process resulting in the efficient removal of most impurities (RNA, chromosomal DNA, proteins, and endotoxins).

Calcium Chloride↗