实验仪器使用技术

MicroRNAs (miRNAs) have emerged as important modulators of eukaryotic gene expression through a process called RNA interference (RNAi). Over the last several years, a large amount of work has focused on understanding how miRNAs are expressed and processed to a biologically functional f ...

The intrinsic potency of a hairpin-based silencing trigger is one of the most critical determinants for applications such as the creation of stable knockdown cell lines or transgenic knockdown animals. To facilitate the creation of potent silencing constructs, we systematically in ...

MicroRNAs (miRNAs) are a class of noncoding small RNAs that can regulate gene expression at the posttranscriptional level. To understand how miRNAs function, it is crucial to determine the mRNA targets that are regulated by specific miRNAs. Based on known miRNA:mRNA interactions, miRNA ta ...

Drug and gene delivery to the central nervous system poses significant challenges to basic researchers and clinicians. The blood–brain barrier prevents most substances from reaching the desired target cells. Strategies for RNA interference (RNAi) have been proposed for certain ne ...

Small, noncoding RNAs have proven to be powerful and ubiquitous agents of gene regulation in eukaryotic genomes. Since their initial discovery, several methods have been developed for directly cloning and sequencing these tiny RNA species. These cloning methods are presented along wi ...

The ability to profile the miRNome (global microRNA/miRNA expression levels) accurately has become essential for multiple aspects of biological research. However, this process is complicated by the short length of the mature miRNA, the existence of multiple intermediate miRNA for ...

Real-time quantitative PCR has become a staple technique of most molecular biology laboratories. Configuration of quantitative PCR instruments into 384-well plates has allowed the technology to function as a low-density gene expression array. In this chapter, we present protocols ...

The in situ detection of microRNAs (miRNAs) has been greatly facilitated by several recent technologic advances. These include the locked nucleic acid (LNA)-modified nucleotide, in situ extension of the mature miRNA, and RT in situ PCR amplification of the pre- and pri-microRNA. Furtherm ...

The structural integrity of siRNA carrying fluorescent dyes attached to its different strands can be �monitored through the efficiency of fluorescence resonance energy transfer between the dyes. In this chapter, the experimental details of the construction of dye-labeled siRNA a ...

Neuropeptide–plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Translocation across the plasma membrane may represent a previously unknown mechanism by which neuropeptides can signal i ...

Analysis of real-time movements of peptidergic vesicles in live neurons provides insight into molecular mechanism(s) supporting the activity-dependent secretion of neurotrophins and neuropeptides. We examined the effect of overexpression of exogenous peptides compri ...

Laser capture microdissection (LCM) provides an efficient and precise method for the sampling of single cells or subgroups of cells in heterogeneous tissues such as the brain. We have recently applied LCM coupled with microarray analysis for the examination of gene expression in the hypot ...

In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for mRNA localization in cell organ ...

Measurements of changes in pre-mRNA levels by intron-specific probes are generally accepted as more closely reflecting changes in gene transcription rates than are measurements of mRNA levels by exonic probes. This is, in part, because the pre-mRNAs, which include the primary transcri ...

In situ hybridization has become a routine technique to provide insights into RNA localization. However, different protocols exist for multiple purposes, and it is, therefore, important to clearly define specific needs to choose the most suitable one(s). For instance, in situ hybridiza ...

The study of neuronal connections and neuron to neuron (or neuron to glia) communication is of fundamental importance in understanding brain structure and function. Therefore, ultrastructural investigation by the use of immunocytochemical techniques is a really precious tool to ...

Neuropeptides are particularly suited to comparative and evolutionary studies, since they have been highly conserved during evolution. Based on primary amino-acid structure, neuropeptides can be arranged into families and synthesized as multiple molecular variants. They m ...

G protein-coupled receptors (GPCRs) comprise one of the largest families of transmembrane proteins involved in signal transduction of diverse external stimuli and represent the most successful target class in drug discovery. The availability of genome sequences in the postgeno ...

Recombinant adeno-associated viral (rAAV) vectors can be used to locally or systemically enhance or silence gene expression. They are relatively nonimmunogenic and can transduce dividing and nondividing cells, and different rAAV serotypes may transduce diverse cell types. The ...

This paper describes an experimental approach based on nanotechnology for assessing the chronic actions of short-lived neuropeptides at specific sites of the brain. This methodology combines the advantages of two different techniques: the microinjection of a suspension of pept ...