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DNase Treatment of RNA

Authors: Isabella Dotti, Serena Bonin,

Publish Date: 2011
Volume: , Issue:, Pages: 87-90
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The presence of contaminating genomic DNA (gDNA) in RNA preparations is a frequent cause of false positives in RT-PCR-based assays aimed at gene expression analysis. Sometimes this phenomenon cannot be avoided even when specific precautions in the assay design are taken (e.g. design of intron-spanning primers), for example, when the target mRNA presents pseudogenes at the DNA level. For these reasons, the inclusion of a DNase digestion step is often necessary. In this chapter two alternative methods for RNA treatment with DNase are described. The choice of the most suitable method is largely dependent on the availability of starting RNA. The conventional DNase treatment requires a step of phenol/chloroform. This method is useful when an RNA solution is not pure and large RNA amounts are available, since this treatment causes about 50% RNA loss. The DNase digestion followed by enzyme heat inactivation is particularly suitable when an RNA starting quantity is very low because, theoretically, no further RNA is lost during heat treatment. This method may be very useful when an RNA has been extracted from small biopsies or cytologic specimens.



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