RNase protection assay methods

In general, quantitative gene expression results obtained with microarray analyses correspond well to results obtained using other methods.11 However, quantitative data from microarrays are typically less reliable for genes that are very highly or very poorly expressed.12 In addition, microarrays are much less sensitive than real time PCR and RNAse protection assays.13 However, the ability to analyze most of the mRNA spe-... 

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

Murthy K, Shen SH, Banville D. A sensitive method for detection of mutations—a PCR-based RNase protection assay. DNA Cell Biol 1995 14(1) 87-94. 

RNase protection assay. This is a sensitive method to determine (1) the amount of a specific mRNA present in a complex mixture of mRNA and/ or (2) the sizes of exons which comprise the mRNA of interest. A radioactive DNA or RNA probe (in excess) is allowed to hybridize with a sample of mRNA (for example, total mRNA isolated from tissue), after which the mixture is digested with... 

Fig. 1. (A) In situ localization of CNTFRa mRNA in coronal section of adult mouse brain. Shown is a low-power dark-field photomicrograph of emulsion-dipped slides of mouse sections hybridized with CNTFRa antisense " S-labeled RNA probe. (B) List of reported distribution of the CNTFRa and method of assessment. Detection of CNTFRa mRNA by Northern Blot, RNase Protection assay or RT-PCR (NRT) or in situ hybridization (ISH). Detection of CNTFRa protein by Western Blot (WB) or immunohistochemistry (IHC). Detection in in vitro primary cell culture (1°).

Assay using relative light units produced by luciferase as the read out by the method described for DNA transfection reporter assay in Subheading 3.3.5. Other methods of choice to determine gene knockdown are Northern blots, RT-PCR, RNase protection, and branched DNA assays, apart from which assays to measure target protein produced can also be used. 

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