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Inhibition of Histone Deacetylases

Lysine acetylation of histones is one of the major epigenetic regulators of chromatin conformation and gene expression. The dynamic nature of histone acetylation is determined by the counterbalancing activity of histone acetyltransferase and histone deacetylase (HDAC) enzy ...

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DNA Methylation Detection: Bisulfite Genomic Sequencing Analysis

DNA methylation, which most commonly occurs at the C5 position of cytosines within CpG dinucleotides, plays a pivotal role in many biological procedures such as gene expression, embryonic development, cellular proliferation, differentiation, and chromosome stability. Aberr ...

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miRBase:The MicroRNA Sequence Database

The miRBase Sequence database is the primary repository for published microRNA (miRNA) sequence and annotation data. miRBase provides a user-friendly web interface for miRNA data, allowing the user to search using key words or sequences, trace links to the primary literature referenc ...

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Methodologies for High-Throughput Expression Profiling of MicroRNAs

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression controlling central biological processes. These small, approx 22-nucleotide (nt)-long RNA molecules induce translational suppression when they are imperfectly matched to their target me ...

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Usefulness of the Luciferase Reporter System to Test the Efficacy of siRNA

Small interefence RNA (siRNA) has revamped the technology of gene silencing in cultured mammalian cells after its first demonstration by Tushl et al. 4 yr ago. To circumvent the cost and the inconvenience in identifying a unique siRNA duplex that can quench target gene expression, we devised a rep ...

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In Situ Hybridization as a Tool to Study the Role of MicroRNAs in Plant Development

MicroRNAs (miRNAs) have a vital role in the generation of plant forms through posttranscriptional regulation of the accumulation of developmental regulators. Analysis of their roles requires detailed knowledge of their expression patterns. We describe an in situ hybridization ...

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Cloning MicroRNAs From Mammalian Tissues

MicroRNAs (miRNAs) are ubiquitous regulators of gene expression in plants and ani-mals. Their distinctive structure, as very short RNAs with a 5′-phosphate and 3′-hydroxyl group, has enabled the development of protocols to clone miRNAs. After enrichment of these small molecules by size, s ...

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Methods for Analyzing MicroRNA Expression and Function During Hematopoietic Lineage Differentiation

MicroRNAs (miRNAs), an abundant class of approx 22-nucleotide (nt) small RNAs that control gene expression at the posttranscriptional level, may play important roles during normal hematopoiesis and leukemogenesis. This chapter focuses on the methods and strategies for dissecti ...

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Identifying MicroRNA Regulators of Cell Death in Drosophila

Animal genomes contain on the order of at least hundreds of microRNAs (miRNAs). Although most remain uncharacterized, it is already clear that miRNAs regulate many bio-logical processes. A number of Drosophila miRNAs have been identified as likely cell death regulators, but functions for ...

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MicroRNAs in Human Immunodeficiency Virus-1 Infection

Initially reported for Caenorhabditis elegans, microRNA (miRNA) has been shown to regulate gene expression in plants, flies, and mammals (1,2). Recently, findings of miRNA have been extended to viruses (3,4). Here, we report on our approaches to investigate the role of miRNAs in human immunode ...

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Cloning and Detection of HIV-1-Encoded MicroRNA

MicroRNAs (miRNAs) are 21- to 25-nucleotides (nt) long and interact with messenger RNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi). We have shown that HIV-1 nef double-stranded RNA from AIDS patients who are long-term nonprogressors, inh ...

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Identification of Messenger RNAs and MicroRNAs Associated With Fragile X Mental Retardation Protein

Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of the Fragile X mental retardation protein (FMRP). FMRP, which may regulate translation in neurons, not only associates with specific messenger RNAs (mRNAs) and with microRNAs (miRNAs), but also associa ...

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Downregulation of Human Cdc6 Protein Using a Lentivirus RNA Interference Expression Vector

Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to e ...

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In Vitro Precursor MicroRNA Processing Assays Using Drosophila Schneider-2 Cell Lysates

Recent studies have shown that the microRNA (miRNA) pathway is an evolutionarily conserved endogenous pathway that is important for normal development. Mature miRNAs are excised from precursors in a stepwise process and subsequently incorporated into an RNA-induced silencing c ...

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Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Nu ...

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Isolation and Identification of Gene-Specific MicroRNAs

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian c ...

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Cloning Gene Family Members Using the Polymerase Chain Reaction with Degenerate Oligonucleotide Primers

As more and more genes are cloned and sequenced, it is apparent that nearly all genes are related to other genes. Similar genes are grouped into families, such as the collagen and globin gene families. There are also gene superfamilies. Gene superfamilies are composed of genes that have areas of high ho ...

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Clone Excision Methods for the Lambda ZAP-Based Vectors

The Lambda ZAP� vectors have been designed to allow in vivo excision and recircularization of the cloned insert and phagemid sequences contained within the λ vector (1). Once a Lambda ZAP library is constructed and amplified, putative clones or the λ library itself may be excised into the phagemid f ...

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Isolation of Messenger RNA from Plant Tissues

The starting material for any representative plant cDNA library is a supply of good-quality messenger RNA from the plant tissue of choice. Extraction of RNA can be made from several grams of tissue or as little as 50 mg. However, large samples are generally more representative of the genes expressed in a ...

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Increasing the Average Abundance of Low-Abundance cDNAs by Ordered Subdivision of cDNA Populations

It is estimated that 50,000–100,000 genes are expressed across the cell types of higher eukaryotes. As a consequence of differential expression (either regionally, temporally, or environmentally specific), a large proportion of all transcripts (approx 40–45%) represent low-abun ...

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