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Proteomic Analysis of Human Mesenchymal Stem Cells

Umbilical cord (UC) and placenta (P) are generally believed to be potential alternatives to bone marrow (BM), as sources of mesenchymal stem cells (MSC) for cell therapy. They possess immunophenotypic and functional characteristics which are similar to that of BM-MSC, yet one of the crucial fa ...

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Metabolic Labeling and Click Chemistry Detection of Glycoprotein Markers of Mesenchymal Stem Cell Differentiation

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into a variety of cell types in vitro including osteoblasts, adipocytes, and chondrocytes. Here we apply a metabolic labeling approach to characterize changes in cellular glycoprotein express ...

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BacMam-Mediated Gene Delivery into Multipotent Mesenchymal Stromal Cells

Baculoviruses have been used over the last several decades for high-level protein production in insect cells. Recently, modified baculovirus containing a mammalian promoter, known as BacMam virus, has been shown to give high transduction efficiencies across several cell types with ...

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Mimicry Technology: Suppressing Small RNA Activity in Plants

Small RNA suppression constitutes one of the major difficulties for a full molecular characterization of their specific roles in plants. Taking advantage of the latest insights into the new post-biogenesis layer of regulation in microRNA (miRNA) activity, it is possible to overcome the a ...

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Experimental Validation of MicroRNA Targets Using a Luciferase Reporter System

MicroRNAs (miRNAs) are a class of small noncoding transcripts that repress gene expression by pairing with their target messenger RNAs (mRNAs). The human genome codes for hundreds of different miRNAs and it is predicted that they target thousands of mRNAs involved in a wide variety of physiol ...

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Experimental Identification of MicroRNA Targets by Immunoprecipitation of Argonaute Protein Complexes

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that negatively regulate gene expression�. Intensive research during the past decade has established miRNAs as key regulators of many cellular pathways. MiRNAs have also been implicated in a number of diseases including var ...

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A High-Throughput Sequencing-Based Methodology to Identify All Uncapped and Cleaved RNA Molecules in Eukaryotic Genomes

How eukaryotic organisms regulate mRNA levels is a fundamental question in biology. It is clear that the steady-state concentration of RNA in a cell is determined by both the rate of its synthesis and turnover. Most of the early attention was focused on the study of gene transcription, while only rece ...

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Target Validation of Plant microRNAs

microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate mRNAs that result in translational inhibition, mRNA decay, or slicing within the region of complementarity. miRNA processing activity on complementary target mRNAs genera ...

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Comprehensive Identification of miRNA Target Sites in Live Animals

MicroRNAs (miRNAs) are small RNA molecules that posttranscriptionally regulate the expression of protein-coding genes. The mature miRNAs are loaded into Argonaute-containing protein complexes (miRISC, miRNA Induced S  ilencing Complex), and guide these complexes to the 3′ UTR of t ...

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Engineering Elements for Gene Silencing: The Artificial MicroRNAs Technology

Small RNA (sRNA)-mediated gene silencing constitutes a powerful tool for the molecular characterization� of a given gene. The RNAi technology has been largely used for this purpose. This approach is based on the cloning of an inverted repeated fragment of the gene to be silenced. Even when this app ...

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Protocols for Use of Homologous Recombination Gene Targeting to Produce MicroRNA Mutants in Drosophila

MicroRNAs (miRNAs) are noncoding RNA molecules that have come to attract considerable interest for their roles in animal and plant development and disease. One means to study miRNA function in animal development is to create mutations. Use of gene-targeting strategies based on ends-out h ...

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MicroRNA Knock Down by Cholesterol-Conjugated Antisense Oligos in Mouse Organ Culture

Here, we detail a protocol to design and introduce sequence-specific cholesterol-conjugated antisense oligonucleotides into mouse organ culture. We review design principles for “antagomirs”, antisense oligos with a cholesterol-moiety modification at the 3′, and present an o ...

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Detection of Small RNAs and MicroRNAs Using Deep Sequencing Technology

Small RNAs (sRNAs) have made a large impact on many recent scientific discoveries. MicroRNAs (miRNAs) are a type of sRNA molecule and, although usually just 20–22 nucleotides in length, they are potent regulators of gene expression. Therefore, identification of miRNAs and profiling their a ...

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Detection of MicroRNAs in Prostate Cancer Cells by MicroRNA Array

MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression at the posttranscriptional level and play a critical role in many important biological processes and pathological development. In the past few years, miRNAs have been implicated to play an importa ...

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Profiling MicroRNAs by Real-Time PCR

A variety of physiological processes are associated with changes in microRNA (miRNA) expression. Analysis of miRNA has been applied to study normal physiology as well as diseased states including cancer. One major challenge in miRNA research is to accurately and practically determine ...

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Detecting sRNAs by Northern Blotting

Small RNAs (sRNAs) play fundamental roles in modifying the transcriptomes and proteomes of organisms. sRNAs can be classified according to their origin and way of action into classes such as microRNAs, trans-acting siRNAs, heterochromatin siRNAs, Piwi-interacting RNAs, and natural ...

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Detection of microRNAs in Plants by In Situ Hybridisation

MicroRNAs (miRNAs) are short, about 21 nucleotides in length, non-coding, regulatory RNA molecules representing a new layer in post-transcriptional gene expression regulation. Spatial and temporal analysis of miRNA accumulation by in situ analyses is the prerequisite of unders ...

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In Situ Detection of microRNAs in Animals

The detection of microRNAs (miRNAs) in situ presents several technical challenges. Although protocols for mRNA detection by in situ hybridization are well established, the small size of miRNAs makes their localization problematic. To overcome this, digoxygenin-labeled locked n ...

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Cell Culture of Human Brain Tumors on Extracellular Matrices: Methodology and Biological Applications

Cell culture is one of the major tools of cell biologists. It has also become an integral part of the daily routine of most oncology laboratories for the purpose of karyotyping, chemoresistance testing, or basic research. It provides investigators with an opportunity to investigate many cell ...

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Isolation and Culture of Human Umbilical Vein Endothelial Cells

The vascular endothelium was, in the past, considered to be relatively pas- sive, merely acting as a filter between the blood and the vessel wall. It is now clear that endothelial cells actively contribute to the maintenance of vascular homeostasis. Endothelial cells synthesize and secrete ac ...

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