Introduction to RNA Quantification and PCR

Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial distribution of RNA expression. A number of widely used procedures exist for detecting and determining the abundance of a particular mRNA in a total RNA sample. The most popular methods are: Northern blot analysis, Nuclease Protection Assays (NPA), in situ hybridization, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

In theory, each of these techniques can be used to detect specific RNAs and to determine their expression level. However, each method has inherent advantages and/or limitations. In general, Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to simultaneously examine multiple messages. In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression. Northern blotting and nuclease protection assays are also limited by their sensitivity. Ribonuclease protection assay is the most sensitive non-PCR based mRNA detection and quantitation procedure but RT-PCR is significantly more sensitive than this; it is capable of detecting moderately expressed transcripts from a single cell.

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