Validating affymetrix chip results with real time pcr

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Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of m RNA populations may be sufficient to initiate and maintain tumour formation.

Total RNA (100 ng from each fraction) was first amplified using the two step amplification protocol of Affymetrix.It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts.Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation.The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug.The technical advance outlined in this manuscript offers the possibility of new insights into m RNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest.

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