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, only 1-5% of target molecules on a blot hybridize to probe (Vernier et al. (1996) Anal. Biochem. 235: 11-19, and data generated at Ambion).
With ULTRAhyb™ and ULTRAhyb-Oligo Ultrasensitive Hybridization Buffer
, the hybridization reaction approaches completion (Figure 1), 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 detected in hours.
ULTRAhyb™ vs. Standard Hybridization Buffer. High specific activity random primed DNA probes were synthesized using Ambion's Strip-EZ™ DNA Kit. The indicated number of these probe molecules were spotted on the BrightStar-Plus™ Membrane on the left as a reference. The indicated quantities of sense strand RNA target molecules were spotted onto the BrightStar-Plus™ Membranes on the right and hybridized overnight to an excess of DNA probe in either standard hybridization buffer or in ULTRAhyb. Note that the hybridization signals generated with ULTRAhyb are difficult to distinguish from the probe molecules spotted on the left.
ULTRAhyb delivers better sensitivity than hybridization solutions offered by other commercial vendors. Figure 2 shows a comparison of hybridization signals from Northern blots using ULTRAhyb versus two other commercially available high sensitivity hybridization solutions.
Random primed, radiolabeled DNA probes were synthesized for the human ornithine decarboxylase gene using Ambion's DECAprime™ II Kit
. Radiolabeled probe (1 x 106
cpm/ml) was hybridized to 50 ng, 25 ng and 12.5 ng of poly(A) RNA from HeLa cells. Hybridizations were done in ULTRAhyb or in hybridization buffers from two other vendors (Figure 2). The manufacturers' recommended procedures were used for the hybridization and wash protocols. Blots were exposed to film for 60 hr at room temperature. The signal obtained with ULTRAhyb was at least 3-5 fold greater than signal obtained using the hybridization buffers from the other vendors.
Comparing Hybridization Buffers. Poly(A) RNA was isolated from HeLa cells. Triplicate RNA samples of 50, 25 and 12.5 ng were electrophoresed on a denaturing agarose gel and the nucleic acid was then transferred to a positively charged nylon membrane. The blot was divided into 3 pieces and hybridized with either: Ambion's ULTRAhyb™ or two other manufacturers' hybridization solutions. The manufacturers' recommended protocols were used for the hybridization and wash procedures. Random-primed radiolabeled probes were synthesized for the human ornithine decarboxylase gene using Ambion's DECAprime™ II Random-Primed DNA Labeling Kit. 1 x 106
cpm/ml of probe was used for each hybridization. Blots were exposed to the same piece of film for 60 hr at room temperature. PerfectHyb is a trademark of Sigma Genosys. MiracleHyb is a trademark of Stratagene.