Titlebook: RT-PCR Protocols; Joe O’Connell Book 20021st edition Humana Press 2002

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Book 20021st editiont. With the advent of PCR, it became possible to amplify specific mRNA, after first converting the mRNA to cDNA via reverse transcriptase. The arrival of this technique—termed reverse transcription-PCR (RT-PCR)—meant that mRNA suddenly became amenable to rapid and sensitive analysis, without the nee

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Rapid Development of a Quantitative-Competitive (qc) RT-PCR Assay Using a Composite Primer Approachnd a shift in the equilibrium of template denaturation, which favors association rather than denaturation of the template DNA strands as product concentration becomes high. This results in a “plateau effect,” such that there is no linearity in the relationship between product yield and initial template.

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Quantitation of Gene Expression by RT-PCR and HPLC Analysis of PCR Productsuantitation (.). Recently, real-time PCR, in which the generated PCR-products are quantified fluorimetrically after each cycle, has become widely used (.). However, determination of fragment size for positive fragment identification is not—at least not directly—possible with this method.

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RT-PCR in Biomedicineechnique permits specific mRNA to be detected and quantified has been a major asset in the molecular investigation of disease pathogenesis. Disease-related imbalances in the expression of specific mRNAs can be sensitively and quantitatively determined by RT-PCR. RT-PCR also offers many opportunities

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The Basics of RT-PCRe fundamental considerations for such basics as primer design, good laboratory set up and practice, and techniques for performing RT-PCR have been fully examined elsewhere (.,.). Thus, I will include only observations from my own experience in this chapter. Although the purpose of this chapter is to

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Nested RT-PCRed by performing “nested” RT-PCR. This involves taking an aliquot of the product from the primary RT-PCR, and using it as a template for a secondary round of PCR amplification. To avoid further amplification of primer-dimer artifacts or nonspecific products generated in the primary PCR, a different

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Quantitative RT-PCRost sensitive technique available for mRNA detection and quantification. It can accurately quantify genes present at only a few hundred copies per sample, and has become the method of choice for the examination of gene expression. The technique consists of two parts: synthesis of cDNA from RNA by re

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