Northern analysis remains a popular technique
Despite the advent of microarrays, Northern analysis remains a popular technique for analyzing gene expression. It is one of the best methods available for evaluating an RNA sample for both quality and quantity. Northerns can also reveal message size and the presence of alternatively spliced transcripts. However, a significant drawback of the technique is that it is relatively insensitive, especially when trying to detect rare targets with standard hybridization buffers, as typically only 1-5% of target molecules on a blot hybridize to probe (Vernier et al. (1996) Anal. Biochem. 235: 11–19). Here we reproduce this standard hybridization buffer data and show that by selecting a more sensitive hybridization buffer, the number of target molecules that hybridize to probe on a blot can be brought closer to 100%.
The blot on the left reveals how much signal results from 1–30x105 randomly-primed DNA probe molecules alone. The blot on the right displays signal resulting from the hybridization of probe to a known amount of target. Comparing the two blots shows what percent of target moleucles are bound to probe. In this example, the signal for 3x106 copies of target is about one-third as intense as 105 copes of probe suggesting that only 1% of the available target is bound by probe in standard hybridization buffer. In ULTRAhyb buffer the signal from 105 copies of target is almost as intense as 105 copies of probe, indicating that most of the target is bound by probe. In fact analyses indicate that as few as 10,000 molecules can be detected on a blot using either RNA or DNA probes (data not shown).