Comparison of linear and exponential protocols using moderate amplifications

Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT) and polymerase chain reaction (PCR), the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. We thus aimed at characterizing deviating gene expressions due to amplification defaults and further checked that differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions) and relevant (biologically validated on unamplified material) Keywords: Amplification, in vitro transcription (IVT), polymerase chain reaction (PCR), biases, molecular features of the affected sequences, relevance of differential expressions, biological validation 96 samples - Amplified material from each tissue (brain, ovary, ovoid embryos, tubular embryos and filamentous embryos) was indirectly labelled using "random" hexamers. Three or two independent targets for each tissue and each protocol (target replicates) were thus generated and hybridised to 4 replicates of the same array (array replicates), so that 48 measurements per probe were generated for somatic (3 targets x 2 tissues x 2 protocols x 4 arrays) and embryonic samples (2 targets x 3 tissues x 2 protocols x 4 arrays).