Simplified high throughput protocol for Northern hybridization

The quantitative determination of a particular mRNA species' concentration within a complex population of transcripts is most readily achieved by Northern blot hybridization analysis. In the most widely employed protocol, RNA samples are fractionated by denaturing agarose gel electrophoresis, transferred to a solid support matrix, and hybridized to radiolabelled complementary probe fragments in the presence of formamide at slightly acidic pH. Historically, the inclusion of formamide was rationalized by the need to lower the annealing temperature required for the formation of probe:target duplexes and preserve the integrity of the RNA which would otherwise be compromised at high temperature, especially at neutral or alkaline pH. Although advantageous in this regard, formamide is not without shortcomings. By retarding the rate of duplex formation formamide limits the sensitivity of target site detection. Moreover, this denaturant is unstable, volatile and significantly toxic. We report here that these limitations are overcome if hybridizations are conducted in a modified version of the tripartite buffer (henceforth referred to as HYBSOL) recently described by Budowle and Baechtel for forensic DNA typing. This approach was used to monitor the induction of tumour necrosis factor alpha (TNF alpha ) and interleukin 1 alpha (IL-1 alpha ) gene expression in rat alveolar macrophages (RAMs) following in vitro exposure to bacterial lipopolysaccharide (LPS) a recognized stimulator of IL-1 and TNF release in this system.