遗传学实验技术

Recently, microRNAs (miRNAs) have been linked to a variety of human diseases including cancer and viral infections. Small molecule modifiers of miRNAs could represent new therapeutic agents and be used as tools for elucidating the biological roles of miRNAs. In order to identify small mole ...

Quantification of microRNAs (miRNAs) in cells or primary tissues is one of the most important steps in elucidating their biological functions. However, miRNAs are challenging molecules for PCR amplification due to the stable hairpin of the precursor form and the small size of the mature miR ...

The therapeutic importance of microRNA (miRNA) regulation has recently been realized as these small, noncoding RNAs have been demonstrated to be involved with a plethora of diseases and disorders. Due to the complex miRNA maturation process, the expression of these important biomolec ...

Numerous studies describe alterations in the levels of specific microRNAs (miRNAs) that are associated with human pathologies. Some of these alterations may give rise to the development of novel diagnostic tools, while certain miRNAs additionally could serve as novel drug targets. Mo ...

MicroRNAs (miRNAs) are a novel class of 17–23 nucleotide short, nonprotein-coding RNA molecules which have emerged to be key players in posttranscriptional gene regulation. In this chapter we give an in-depth review of the classic, canonical mammalian miRNA maturation pathway and disc ...

RNAs need to adopt a specific architecture to exert their task in cells. While significant progress has been made in describing RNA folding landscapes in vitro, understanding intracellular RNA structure formation is still in its infancy. This is in part due to the complex nature of the cellular e ...

In this chapter we describe the use of two methods, in-line probing as well as terbium(III) cleavage. Both methods can be applied to RNAs of any size, structure, and function. Aside from revealing directly metal ion-binding sites these techniques also provide structural information for longer ...

In recent years RNA molecules have emerged as central players in the regulation of gene expression. Many of these noncoding RNAs possess well-defined, complex, three-dimensional structures which are essential for their biological function. In this context, much effort has been devot ...

Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide’s structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of m ...

RNAs have to adopt specific three-dimensional structures to fulfill their biological functions. Therefore exploring RNA structure is of interest to understand RNA-dependent processes. Chemical probing in vitro is a very powerful tool to investigate RNA molecules under a variety of ...

Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecule’s reactivity to different probes is quantified at single nucleotide resolution and used to constrain structural modeling. This experimental framework has been extensively re ...

Enzymatic probing is a rapid, straightforward method for determining which regions of a folded RNA are structurally constrained. It can be carried out using very small amounts of material, and is especially suitable for short RNAs. Here we report a protocol that we have found to be useful and readily ...

RNA-mediated biological processes usually require precise definition of 5′ and 3′ ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with ...

RNA secondary structures can be predicted using efficient algorithms. A widely used software package implementing a large number of computational methods is the ViennaRNA Package. This chapter describes how to use programs from the ViennaRNA Package to perform common tasks such as pre ...

RNA forms defined structures and binds specifically to target molecules. The combination of data which results from fluorescence correlation spectroscopy (FCS) and small angle X-ray scattering (SAXS) measurements can be used to determine intermolecular interactions between ...

Conformational transitions (refolding) between long-lived conformational states constitute the time-limiting step during the folding process of large RNAs. As the dynamics of these reactions dominate the regulatory and other functional behavior of RNA molecules, it is of impo ...

RNA folding pathways can be complex and even include kinetic traps or misfolded intermediates that can be slow to resolve. Characterizing these pathways is critical to understanding how RNA molecules acquire their biological function. We have previously developed a novel approach to h ...

Analytical Ultracentrifugation (AUC) is a highly sensitive technique for detecting global conformational features of biological molecules and molecular interactions in solution. When operated in a sedimentation velocity (SV) recording mode, it reports precisely on the hyd ...

The study of biologically relevant native RNA structures is important to understand their cellular function(s). Native gel electrophoresis provides information about such native structures in solution as a function of experimental conditions. The application of native gel ele ...

Here two methods to measure RNA chaperone activity in vivo and in vitro are described. In both assays folding of a misfolded group I intron RNA into the splicing-competent form in the presence of proteins with RNA chaperone activity is measured. Folding is evaluated indirectly by assessing the di ...