Northern Analysis: Faster and Better

•Increase the sensitivity of any blot hybridization
•Maximize signal without increasing background
•Decrease hybridization and exposure time
•Flexible -- use DNA or RNA probes labeled isotopically or nonisotopically

Using standard hybridization buffers only 1-5% of target molecules on a blot hybridize to probe, making Northern blotting a relatively insensitive method for nucleic acid analysis (Vernier 1996, and data generated at Ambion). With ULTRAhyb® Ultrasensitive Hybridization Buffer the hybridization reaction approaches completion, so that as few as 10,000 molecules can be detected. ULTRAhyb contains a unique blend of hybridization accelerators and blocking agents that greatly enhance the levels of hybridization, so that signals that once took days to visualize are now apparent in hours. In Figure 1, replicate Northern blots were prepared with mouse thymus total RNA and hybridized with a ³²P-labeled DNA probe complementary to p53. Hybridizations were performed with various hybridization solutions using the indicated protocols. p53 was detected in 0.5 µg total RNA in a 4 hour exposure using ULTRAhyb, while the other solutions tested required 2 µg RNA (4 times more target) and 40 hours exposure (10 times more exposure time) to produce similar levels of signal. ULTRAhyb increases sensitivity; many messages can be detected with only a 2 hour hybridization. In general, if a message is visible in an overnight hybridization using standard hybridization buffers, it will be visible with a mere 2 hour hybridization using ULTRAhyb.

Figure 1. ULTRAhyb Detects a Signal in Only 8 Hours, Compared to a Minimum of 5 days Using a Competitor’s Hybridization Solution. Replicate Northern blots loaded with 2.0 or 0.5 µg mouse thymus total RNA were assayed for p53 using 106 cpm/ml of a random prime labeled DNA probe. The blots were incubated in the hybridization buffers indicated following the manufacturer's recommendations for time and temperature. All blots were washed using 2X SSC/0.1% SDS and 0.1X SSC/0.1% SDS. The blots were exposed to the same piece of film at -80°C with one intensifying screen for the indicated times.

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