Ultrasensitive Hybridization Buffer | Detect Rare Transcripts
|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). |
Compare and Get Higher Sensitivity
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.