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Northern Analysis: The Basics

.g., cDNA from a different species or genomic DNA fragments that might contain an intron) may be used as probes.

Despite these advantages, there are limitations associated with Northern analysis. First, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. Second, a standard Northern procedure is, in general, less sensitive than nuclease protection assays and RT-PCR, although improvements in sensitivity can be achieved by using high specific activity antisense RNA probes, optimized hybridization buffers and positively charged nylon membranes. Sensitivity can be further improved with oligo dT selection for enrichment of mRNA, since physical constraints of gel electrophoresis and membrane transfer limit the amount of RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax reagents in combination with ULTRAhyb (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of Northern blotting has been the difficulty associated with multiple probe analysis. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second
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