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Northern Analysis: The Basics

probes, and that RNA probes provide an additional 10-fold increase in sensitivity. RNA probes have the added advantage that they can be hybridized and washed under more stringent conditions, which results in lower background and fewer problems with cross-hybridization. For these reasons, Ambion has long been a proponent of using RNA probes for assaying Northern blots, although the use of ULTRAhyb Ultrasensitive Hybridization Buffer (see below) drastically reduces the disparity in sensitivity between RNA and DNA probes. Both types of probes can detect fewer than 100,000 molecules on a blot with ULTRAhyb, making the decision to use RNA or DNA probes primarily a matter of preference for a particular labeling technique.

RNA probes can be produced by in vitro transcription reactions using Ambion's MAXIscript or Strip-EZ RNA Kits. Strip-EZ RNA probes include a modification that allows them to be easily removed or "stripped" off blots, in contrast to standard RNA probes. Isotopic or nonisotopic labeled nucleotides can be incorporated directly during synthesis with either kit, or the RNA can be synthesized unlabeled and subsequently treated with Psoralen-Biotin to produce biotinylated probes for blot hybridizations. For more information about using RNA probes in Northern analysis, see "'"/>

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