Abstract
BackgroundSSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA.ResultsFour independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs) tester-specific transcripts.ConclusionThe close agreement between our hybridization and sequencing results shows that the addition and follow-up of exogenous reporter transcripts provides an easy and reliable means to check SSH performance. Despite some cases of irregular normalisation and subtraction failure, we have shown that SSH repeatedly enriches the libraries in very rare, tester-specific transcripts, and can thus be considered as a powerful tool to investigate situations where small amounts of biological material are available, such as during early mammalian development.
Highlights
Suppression Subtractive Hybridisation (SSH) has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations
In order to achieve a broad appraisal of the quality of the libraries, we decided to array several thousand clones from each library and analyse the abundance of tester-specific and tester/driver common transcripts in the subtracted libraries after bacteria transformation
Neither strict tester-specific transcripts nor tester/driver common and expressed transcripts are identified at these stages in bovine and rabbit embryos, so we introduced exogenous A. thaliana transcripts into our biological material prior to pre-amplification and subtraction
Summary
SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. Technical improvements in the analysis of messenger RNAs from tiny amounts of cells have revealed the complexity of the genome expressed during the preimplantation period [1,2] This complexity has been highlighted in recent publications which reported the isolation of new sequences in different mammalian species [3,4,5,6]. Because SSH does not require previous knowledge of gene sequences, it may be suitable for species where only a small number of sequences are available in databases
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