TRANSCRIPTOME 2002: From Functional Genomics to Systems
The aRNA Amplification Procedure in mRNA-Poor Samples Array Applications
Esther Graudens, Xavier Barlet, Virginie el Marhomy, Charles Auffray & Sandrine Imbeaud, Array s/IMAGE, Genexpress team, Villejuif Cedex, FRANCE
For several years, the Genexpress has investigated transcriptomes through high-throughput gene expression profiling developing cDNA array approaches. One important limitation is the requirement for large amounts of RNA for array hybridization, typically 10-20µg of total RNA. This is especially a problem when working with limited samples such as laser capture microdissection. Our team is focusing high-attention on the optimization of the array procedures in term of reduced sample consumption keeping data precision. The aRNA amplification is a procedure we used in mRNA-poor samples programs. It consists of reverse transcription with a T7 promoter-oligo(dT) and in vitro transcription of the resulting cDNA to generate antisense RNA copies of each mRNA in a sample. In our hand, a single reaction, starting with 1µg total RNA (around 10 ng mRNA) yields a minimum of 10µg aRNA (e.g. 1000x amplification). We show the aRNA synthesis being tightly dependent on the efficiency and fidelity of such complex enzymatic reactions. We identify the primer sequence itself as a critical factor and report the analysis of three different ones, testing the influence of either the dT size or T7 sequence. Characterization of the cDNA and aRNA has been done using UV measurement, Agilent LabChip and array hybridization.
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