基因表达差异显示实验

Most metazoan genes are alternatively spliced, and a large number of alternatively spliced isoforms are likely to be functionally significant and expressed at specific stages of pathogenesis or differentiation. Splicing changes usually only affect a small portion of a gene, and these ...

RNA localization is a cellular process to spatially restrict translation of specific proteins to defined regions within or between cells. Most localized mRNAs contain cis-acting localization elements in the 3′-untranslated region (UTR), which are sufficient for localization of an ...

All eukaryotic cells display a dramatic partitioning of mRNAs between the cytosol and endoplasmic reticulum (ER) compartments—mRNAs encoding secretory and integral membrane proteins are highly enriched on ER-bound ribosomes and mRNAs encoding cytoplasmic/nucleoplasm ...

Regulating gene expression at the translational level controls a wide variety of biological events such as development, long-term memory, stress response, transport and storage of certain nutrients, and viral infection. Protein synthesis at steady-state level can be directly mea ...

The number and distribution of ribosomes on a transcript provide useful information in ascertaining the efficiency of translation. Herein we describe a direct method to determine the association of ribosomes with specific regions of an mRNA. The method, termed Ribosome Density Mappi ...

The inability of structural elements within a reporter mRNA to impede processive decay by the major 5′ and 3′ exonucleases has been a major obstacle to understanding mechanisms of vertebrate mRNA decay. We present here a new approach to this problem focused on quantifying the decay of individual ...

Sequence elements within mRNA-untranslated regions and their binding partners are key controllers of mRNA stability. Changes in mRNA stability can often be detected by changes in steady-state mRNA abundance, or a more careful analysis of mRNA half-lives can be performed following tra ...

Cytoplasmic extracts have proven to be a versatile system for assaying the mechanisms and interactions of RNA metabolism. Using Aedes albopictus (C6/36) cells adapted to suspension culture, we have been able to faithfully reproduce and manipulate all aspects of mRNA decay in vitro. Descr ...

IMGT/V-QUEST is the highly customized and integrated online IMGT� tool for the standardized analysis of the immunoglobulin (IG) or antibody and T cell receptor (TR) rearranged nucleotide sequences. The analysis of these antigen receptors represents a crucial challenge for the study of t ...

DNA methylation of retrotransposons and imprinted genes is accurately regulated in spermatogenesis. In particular, CpG methylation of long interspersed elements-1 (LINE1 or L1) and intracisternal A-particle (IAP) retrotransposons during spermatogenesis has been well ch ...

Piwi-interacting RNAs (piRNA) are fully modified by 2′-O-methylation at their 3′-termini. This terminal methylation is required to prevent 3′-nucleotide addition, which serves as a tag for destabilization. In this chapter, we describe biochemical and mass spectrometric analyses of 2 ...

Piwi proteins and their small-RNA partners, piwi-interacting (pi)RNA, form a natural mechanism that prevents the deleterious activity of transposable elements in the germ line of metazoan species. The piRNA pathway relies on extended noncoding genomic regions, dubbed piRNA clust ...

Classical biochemical approaches have made great contribution to our current understanding of how small interfering RNA (siRNA) and microRNA (miRNA) are produced and function. However, it has been challenging to establish in vitro systems that can dissect the mechanism of PIWI-inte ...

Emerging evidence indicates that PIWI proteins, in collaboration with PIWI-interacting RNAs (piRNAs), play a critical role in retrotransposon silencing in Drosophila gonadal somatic and germ-line cells. The recent establishment of female germ-line stem cells/ovarian somat ...

The Drosophila piwi gene has multiple functions in soma and germ cells. An in vitro system provides a powerful tool for elucidating PIWI function in each cell type using stable cell lines originating from germline stem cells (GSCs) and ovarian soma of adult ovaries. We have described methods for the ...

The generation of high-resolution maps of the epigenome is crucial to research in epigenetics, developmental biology, and stem cell biology. In recent years, small RNA pathways have been implicated in epigenetic regulation. All small RNA pathways involve Argonaute proteins as their key ...

The small noncoding RNAs in plants are categorized into two major classes, 21-nucleotides (nt) micro RNA (miRNA) and 21- or 24-nt small-interfering RNA (siRNA). ARGONAUTE (AGO) proteins associate with small RNAs and play central roles in transcriptional and posttranscriptional gene re ...

Gene knockdown using double-stranded RNA (dsRNA) is a powerful tool to characterize gene function in Drosophila. The Drosophila germline provides an elegant model to study the regulation of numerous processes, such as stem cell fate, gametogenesis, piRNA, and piRNA-related gene funct ...

Piwi proteins and piRNAs are abundant in the gonads of various animal species. Gonads from different developmental stages provide us information regarding the function of piRNAs and the PIWI pathway during germline development. Here we describe methods for gonad and germ cell preparat ...

The recent development of High Throughput Sequencing technology has boosted the study of small regulatory RNA populations. A critical step prior to cloning and sequencing is purification of small RNA populations. Here, we report the optimization of an anion-exchange chromatography ...