Reverse transcription-PCR differential display analysis of Escherichia coli global gene regulation in response to heat shock

A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli. A novel combination of primers designe d specifically for the start and stop regions of E. coli genes (based on th e findings of Fislage et al. [R, Fislage, M, Berceanu, Y, Humboldt, M. Wend t, and N. Oberender, Nucleic Acids Res. 25:1830-1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The va lidity of the technique was demonstrated by applying it to heat shock analy sis, Specifically, RT-PCR-amplified total RNA from heat-shocked and non-hea t-shocked cells were hybridized with slot blots of the Kohara set (U, Kohar a, K, Akiyama, and K. Isono, Cell 50:495-508, 1987; S. Chuang, D, Daniels, and F. Blattner, J, Bacteriol. 175:2026-2036, 1993), The signals obtained f or heat-shocked and control cultures of each clone were compared, and diffe rences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation, A lso, for several genes, Northern blotting and total RNA dot blotting were p erformed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for st udying global gene regulation in E, coli by incorporating a PCR amplificati on step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study E. coli global gene re gulation in response to additional environmental stimuli.