Transcriptome Subtractive Hybridization and its Reliability Validated by an E. coli Model

In the application of subtractive hybridization methods to identify differentially expressed genes, It is no doubt that would increase the efficiency of subtractive hybridization method by simplifying experimental procedures and reducing experimental time-consuming. In this study, we reported a modified method, called transcriptome subtractive hybridization (abbr. TSH). In order to cover different types of RNA as much as possible and reduce the experimental procedure, TSH in the protocol neither uses restriction endonuclease for adopter ligation, nor use oligo-d (T) for the reverse transcription reaction. Instead, tester RNA directly hybridizes with driver single strand cDNA, then the hybrid of RNA/cDNA was digested by Hae III, residual targeting tester RNA was enriched by reverse transcription PCR amplification. As a result, the experimental protocol was simplified to reduce experimental time-consuming. To validate use reliability of the TSH, TSH was employed to identify cell-specific expressing ESTs in both E. coli JM109 with and without a recombinant plasmid. The results showed that the non-target RNA finally eliminated through direct hybridization and digestion of RNA/cDNA hybrids. Six reference genes within the recombinant plasmid were detected as expected in the tester library constructed from the JM109 RNA with the recombinant plasmid. Obviously, the verified results showed that the TSH, a modified protocol, would be a reliable and effective method, which can be applied to identify differential gene expression between the two cell types.