细胞技术

The purity and integrity of Isolated RNA IS a critical determinant of its effectiveness in such molecular biological procedures as Northern blot, poly A+ RNA separation, cDNA synthesis, and in vitro transcription and translation The successful isolation of total RNA from tissues or cell li ...

The susceptibihty of RNA to degradation by exogenous and endogenous RNase activity following cell lysis has been well documented (1,2). Moreover RNA usually occurs complexed with protein from which it must be released. Precautions to be taken against exogenous RNase include the use of plas ...

Whole blood contains nucleated white cells that constitute an easily accessible source from which RNA can be extracted, without the need for prior homogemzation as is necessary with solid tissues. However, blood is a particularly problematic tissue from which to isolate RNA because RNA is e ...

In plant cells, mitochondrial RNA (mtRNA) constitutes about only 1% of the total RNA. From this, most are ribosomal RNAs. Thus, isolation of high-purified mtRNA is necessary not only for construction of a mitochondrial cDNA library, but also for the analysis of plant mitochondrial transcript ...

The isolation of uncontaminated, intact RNA is essential for analyzing gene expression and for cloning genes. However, plant tissue is notorious for being a difficult source from which to isolate high-quality RNA with good yield. This difficulty is primarily due to the presence of naturally ...

Fixed and paraffin-embedded tissues from pathology department archives can be available for RNA expression analysis. We have already shown that RNA isolated from biopsy, surgical, or autopsy tissue, routinely processed by fixation and paraffin embedding, is not completely degrad ...

Cationic surfactants precipitate nucleic acids, presumably by forming reverse micelles in which the ahphatic tails of the surfactant face the aqueous phase, and the cationic head groups bind to the nucleic acid electrostatically. The precipitate can be dissolved in organic solvents, ...

With the introduction of polymerase chain reaction (PCR), analysis on even minute amounts of DNA and RNA from biological samples became practicable. Conventional methods of mRNA analysis are often not sensitive enough for detection of low-abundance transcripts or broad examination ...

Changes in cell behavior are driven by changes in gene expression. Thus, in order to understand the mechanisms regulating cell behavior, one has to identify and characterize differentially expressed genes. Standard methods currently used to isolate differentially expressed genes ...

The purification of good-quality RNA from tissue-cultured cells is essential for many applications and several methods exist for the isolation of total RNA. Most protocols rely on sodium dodecyl sulfate (SDS) (1) or guamdium thiocyanate (2,3) to simultaneously lyse cells and mactivate e ...

From the very beginning of PCR technology (1– 3), it was clear that the power of amplification could be used for the study of mRNA expression (4,5). This technique is now wldeiy used and many different protocols have been developed using either viral reverse transcriptases (RT) or exploiting the reve ...

Nonradioactively labeled probes offer several advantages compared to radioactive ones, as they show long stability, high morphologrcal resolution, and rapid developing time. There are different types of nonradioactive labeling methods available, although dlgoxigenin- ...

Methods allowing sensitive and accurate quantitative analysis of defined RNA species are required in a wide variety of gene expression studies. Unlike the traditional hybridization methods, RNase protection or S1 nuclease assays (see Chapters 16 and 27), the methods based on reverse tr ...

As already reported in a previous chapter of this book, it is possible to extract and analyze RNA from fixed- and paraffin-embedded tissues (see Chapter 5). For many purposes, a simple qualitative analysis of the presence of a specific RNA in tissues may be sufficient, for instance, to establish the per ...

Adenosine 3′,5′-cyclic monophosphate (cAMP) is involved in a myriad of normal and pathological processes. Indeed, this cyclic nucleotide serves as a second messenger for the action of endogenous and exogenous agents in organisms ranging from bacteria to humans (1). The ubiquitous nature ...

The generation of cyclic AMP (cAMP) is probably the most scrutinized of signal transduction pathways. Receptors may be linked to the generation of AMP by one of two routes. One group of receptors (such as β-adrenoceptors, A2 adenosine receptors, D1 dopamine receptors, histamine H2 receptors, a ...

In mammals, adenylyl cyclase is a family of membrane-bound enzymes that catalyze the conversion of adenosine triphosphate (ATP) to adenosine 3′:5′-cyclic monophosphate (cAMP). cAMP is an ubiquitous intracellular signaling molecule that modifies cell function by activating cAMP ...

The methods described in this chapter are designed to measure the hydrolysis of guanosine triphosphate (GTP) to inorganic phosphate (Pi) and guanosine diphosphate (GDP), a reaction catalyzed by the GTPase enzymes (EC 3.6.1.-). The theory behind the experimental design involves using GTP as a ...

Nitric oxide (NO) is an important biological mediator involved in control of blood vessel tone, neurotransmission, and immune function (1–3). NO has physicochemical properties that make it ideal to function as an intercell communicator, and these include an ability to travel rapidly bet ...

Phospholipases A2 (PLA2s) are important enzymes in signal transduction, being responsible for release of arachidonate from membrane phospholipids for the production of prostaglandins, leukotrienes, platelet-activating factors, and other bioactive lipids (1). Phospho ...