MULTI-SAMPLE COMPARATIVE RT-PCR

Ambion, Inc. proposes to adapt its Comparative RT-PCR procedure to accommodate the comparative analysis of single transcripts across one hundred RNA samples. The proposed procedure involves reverse transcribing a collection of RNA samples using primers with identical anchored oligodT and PCR primer binding sites but intervening hybridization domains that are unique to each sample. Equal amounts of the differentially tagged cDNAs are mixed and a specific target sequence in the sample mixture is amplified using a primer specific to the binding site present in the reverse transcription primer and a primer specific to the target of interest. A labeled nucleotide or primer is incorporated during PCR and the labeled products are hybridized to an array of probes specific to each of the sample-specific hybridization domains. The hybridization signal is quantified to reveal the relative abundance of the target in the RNA samples. The cDNA mixture can be used to evaluate many unique genes.