RNase protection assays

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-... 

Sassone-Corsi In the experiment you showed it is clear that in all those receptors there is extensive alternative splicing. In an RNAse protection assay, picking the probe is crucial. 

Figure 5.8 Detection of PNA-induced 7-globin gene expression in K562 cells with RNase Protection Assay. Lane 1, K562 cells lane 2 to lane 4, K562 cells treated with PNAs for two, three, and four days, respectively lane 5, K562 cells treated with hemin (75 pM) for three days.

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. 

Downstream application [PCR (polymerase chain reaction), cloning, labeling, blotting, RT (reverse transcriptase)-PCR, cDNA synthesis, RNAse protection assays, gene therapy, etc.]... 

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... 

SI end mapping. A technique to determine where the end of an RNA transcript lies with respect to its template DNA (the gene). Can t be described in a short paragraph. See RNAse Protection assay for a closely related technique. 

RNase protection assays are very powerful but more complex to perform than Northern blots. In this case an RNA probe that is complementary to the RNA of... 

SI analysis is widely used to determine the ends of an mRNA molecule, the sites of introns or to quantify mRNA. Recently, RNase protection assays have become increasingly popular since they are more sensitive and can be used to obtain the same information. However, background is much less of a problem in SI analysis. The... 

RNase protection assays (RPA) are based on the property of RNase to digest ii RNA, but not ds RNA, and its principles and applications resemble those of SI analysis (Lynn et al., 1983 Zinn et at, 1983) (Fig. 12.3). The sequence of interest is inserted in a plasmid downstream of a bacteriophage promoter (e.g., pUC118, pT7, etc.. Table 4.4). The purified plasmid is then restricted downstream of the inserted DNA and the linearized plasmid is transcribed in the presence of a labeled rNTP precursor. The transcript should be complementary to the RNA to be studied and an excess of probe is hybridized to its target. Any RNA remaining ss is then digested by one or more RNases. The size of the RNase-resistant probe and the... 

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