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