Biological Procedures Online

https://biologicalproceduresonline.biomedcentral.com

List of Papers (Total 314)

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure...

Experimental assessment of the role of acetaldehyde in alcoholic cardiomyopathy

Alcoholism is one of the major causes of non-ischemic heart damage. The myopathic state of the heart due to alcohol consumption, namely alcoholic cardiomyopathy, is manifested by cardiac hypertrophy, compromised ventricular contractility and cardiac output. Several mechanisms have been postulated for alcoholic cardiomyopathy including oxidative damage, accumulation of...

Methodology for detecting trace amounts of microchimeric DNA from peripheral murine white blood cells by real-time PCR

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report...

Negative staining and image classification — powerful tools in modern electron microscopy

Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image...

Ribosome formation from subunits studied by stopped-flow and rayleigh light scattering

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for...

New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell...

Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR

Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays...

An Alternative Model of Laser-Induced Stroke in the Motor Cortex of Rats

BackgroundA common experimental rodent model for stroke includes induction by a technique in which middle cerebral artery is transiently (MCAO-t) or permanently (MCAO-p) occluded by catheterization. However, this model has prominent disadvantages which consist of the high variability of localization and size of the ischemic area, cases of intracranial hemorrhage and high...

Use of Plasmid pVMG to Make Transcriptional ß-Glucuronidase Reporter Gene Fusions in the Rhizobium Genome for Monitoring the Expression of Rhizobial Genes In Vivo

BackgroundThe soil bacterium Sinorhizobium meliloti and its allies are important nitrogen-fixing bacterial symbionts that cause N2-fixing nodules on the roots of legumes. Chromosomal ß-glucuronidase gene (uidA) transcriptional fusions are frequently used to monitor the expression of bacterial genes during the symbiosis. However, the construction of the fusions is laborious...

A Cautionary Tale: Endogenous Biotinylated Proteins and Exogenously-Introduced Protein A Cause Antibody-Independent Artefacts in Western Blot Studies of Brain-Derived Proteins

Antibodies are commonly used to detect or isolate proteins from biological samples. Much attention has been paid to the potential for poorly-characterized antibodies to lead to misleading results, but antibody-independent artefacts may also occur. Here, we recount two examples of antibody-independent artefacts that have confounded the interpretation of results in our search for...

Development of Effective Therapeutics Targeting HER3 for Cancer Treatment

HER3 is the third member of the human epidermal growth factor receptor (HER/EGFR) family, and unlike its other family members, is unique due to its minimal intrinsic kinase activity. As a result, HER3 has to interact with another receptor tyrosine kinase (RTK), such as EGFR or HER2, in order to activate the PI-3 K/Akt, MEK/MAPK, Jak/Stat pathways, as well as Src kinase. Over...

Effective Visualization and Easy Tracking of Extracellular Vesicles in Glioma Cells

Extracellular vesicles (EVs) are nano-sized, membrane-bound structures secreted by cells and play critical roles in mediating intercellular signaling. EVs based on their size as well as mechanisms of biosynthesis are categorized as either microvesicles (200–1000 nm) or exosomes (30–200 nm). The EVs carry several biomolecules like proteins, DNAs, RNAs, and lipids into other cells...

A simple and reliable protocol for long-term culture of murine bone marrow stromal (mesenchymal) stem cells that retained their in vitro and in vivo stemness in long-term culture

BackgroundBone marrow derived stromal stem cells (BMSCs) are a clonogenic cell population that is characterized by self-renewal capacity and differentiation potential into osteoblasts, and other mesenchymal cell types. Mouse BMSCs (mBMSCs) are difficult to be cultured and propagated in vitro due to their replicative senescent phenotype, heterogeneity and high contamination with...

Regeneration of TiO2 Nanotube Arrays after Long-Term Cell and Tissue Culture for Multiple Use – an Environmental Scanning Electron Microscopy (ESEM) Survey of Adult Pig Retina and beyond

Long-term organotypic culture of adult tissues not only open up possibilities for studying complex structures of explants in vitro, but also can be employed e.g. to investigate pathological changes, their fingerprints on tissue mechanics, as well as the effectiveness of drugs. While conventional culture methods do not allow for survival times of more than a few days, we have...

Circular Estimate Method (CEM) - a Simple Method to Estimate Caenorhabditis elegans Culture Densities in Liquid Medium

BackgroundNematodes are used in many different fields of science, including environmental and biomedical research. Counting and/or estimating nematode numbers is required during research. Although being one of the most common procedures, this apparently simple task is a time-consuming process, prone to errors and concerns regarding procedure, reliability, and accuracy. When an...

Performance evaluation for MOTIFSIM

BackgroundPrevious studies show various results obtained from different motif finders for an identical dataset. This is largely due to the fact that these tools use different strategies and possess unique features for discovering the motifs. Hence, using multiple tools and methods has been suggested because the motifs commonly reported by them are more likely to be biologically...

Three-Dimensional Quantification of Spheroid Degradation-Dependent Invasion and Invadopodia Formation

Invadopodia are actin-rich, proteolytic structures that enable cancer cell to invade into the surrounding tissues. Several in vitro invasion assays have been used in the literature ranging from directional quantitative assays to complex three-dimensional (3D) analyses. One of the main limitations of these assays is the lack of quantifiable degradation-dependent invasion in a...

Modified SureSelectQXT Target Enrichment Protocol for Illumina Multiplexed Sequencing of FFPE Samples

BackgroundPersonalised medicine is nowadays a major objective in oncology. Molecular characterization of tumours through NGS offers the possibility to find possible therapeutic targets in a time- and cost-effective way. However, the low quality and complexity of FFPE DNA samples bring a series of disadvantages for massive parallel sequencing techniques compared to high-quality...

A New ELISA to Overcome the Pitfalls in Quantification of Recombinant Human Monoclonal Anti-HBs, GC1102, by Commercial Immunoassays

Several methods for the quantification of human anti-HBs, an antibody to hepatitis B surface antigen (HBsAg), have been developed based on enzyme reaction, chemiluminescence, fluorescence, and radioactivity for application to human serum or plasma. Commercial anti-HBs immunoassay kits use a sandwich method in which a bridge is formed by the anti-HBs between a HBsAg immobilized...

microRNA Regulation in Estrogen Receptor-Positive Breast Cancer and Endocrine Therapy

As de novo and acquired resistance to standard first line endocrine therapies is a growing clinical challenge for estrogen receptor-positive (ER+) breast cancer patients, understanding the mechanisms of resistance is critical to develop novel therapeutic strategies to prevent therapeutic resistance and improve patient outcomes. The widespread post-transcriptional regulatory role...

Targeting of HER3 with Functional Cooperative miRNAs Enhances Therapeutic Activity in HER2-Overexpressing Breast Cancer Cells

BackgroundThe HER3 receptor functions as a major cause of drug resistance in cancer treatment. It is believed that therapeutic targeting of HER3 is required to improve patient outcomes. It is not clear whether a novel strategy with two functional cooperative miRNAs would effectively inhibit erbB3 expression and potentiate the anti-proliferative/anti-survival effects of a HER2...

Optimization of Immunofluorescent Detection of Bone Marrow Disseminated Tumor Cells

BackgroundCancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the...

Icariin: a Potential Compound for the Recovery of Tibial Dyschondroplasia Affected Chicken Via Up-Regulating BMP-2 Expression

BackgroundTibial dyschondroplasia (TD) is a skeletal disease of fast growing chicken and other avian species. It is characterized by an avascular and non-mineralized growth plate, which leads to a deformed tibial bone and lameness. Unfortunately, this disease is not only responsible for causing huge economic losses but also raises animal welfare concerns. Icariin is a flavonoid...

Isolation and Semi Quantitative PCR of Na+/H+ Antiporter (SOS1 and NHX) Genes under Salinity Stress in Kochia scoparia

BackgroundKochia scoparia is a dicotyledonous annual herb and belongs to the Amaranthaceae family. Genetic diversity and resistance to drought stress of this plant has made it widely scattered in different regions which contains highly genetic diversity and great potential as fodder and can grow on salty, drought affected areas. Since the soil salinity has become widely spread...

Blockade of ITGA2 Induces Apoptosis and Inhibits Cell Migration in Gastric Cancer

BackgroundGastric cancer is currently the fourth leading cause of cancer-related death worldwide. Gastric cancer is often diagnosed at advanced stages and the outcome of the treatment is often poor. Therefore, identifying new therapeutic targets for this cancer is urgently needed. Integrin alpha 2 (ITGA2) subunit and the beta 1 subunit form a heterodimer for a transmembrane...