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Northern Analysis: The Basics

from coating the membrane. Good blocking is necessary to minimize background problems.

The NorthernMax and NorthernMax-Gly kits include ULTRAhyb Ultrasensitive Hybridization Buffer, which can be used for both prehybridization and hybridization. Although double-stranded DNA probes must be denatured prior to use, RNA probes and single-stranded DNA probes can be diluted in a small amount of ULTRAhyb and then added to the prehybridized blot. ULTRAhyb can increase sensitivity up to 100-fold compared to other hybridization solutions (Figure 2) by pushing hybridization to completion without increasing background. As few as 10,000 molecules can be detected. Because ULTRAhyb maximizes blot sensitivity, hybridization can be performed in just 2 hours for many messages. The largest increase in sensitivity is seen using random-primed DNA probes and ULTRAhyb. Under these conditions, signals are as intense as those seen with RNA probes.

Figure 2. ULTRAhyb versus a standard hybridization buffer using DNA and RNA probes. Identical Northern blots of indicated amounts of mouse thymus total RNA were hybridized overnight with radiolabeled StripAble DNA or RNA probe. The blots were incubated with probe in either ULTRAhyb or standard hybridization buffer as indicated. Standard low and high stringency SSC/SDS washes were used to remove nonspecifically bound probe. Blots were exposed to film for 2.5 hours with a single intensifying screen.


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