Transcriptome Profiling: Amplification of Oligonucleotide Barcodes

We describe a new method to synthesize and amplify cDNA for gene expression profiling on oligonucleotide microarrays. Currently, two methods used to amplify targets are polymerase chain reaction (PCR) or T7 polymerase based in vitro transcription. Conventional PCR due to its non-linear nature is prone to bias in amplification of multiple templates. Although, In Vitro transcription using T7-oligo(dT) or random primers amplify transcripts linearly, it has been reported that they fail to capture complete 5' information of transcripts and detect presence of rare transcripts. Both methods are further prone to cross hybridization effects on microarrays. To address these issues, we have developed a method using gene specific primers to capture information along any region of the gene.

Instead of cDNA amplification, a reporter sequence unique to a particular mRNA transcript is amplified and detected by complementary probe on microarray. The reporter tags (oligonucleotide barcodes) are incorporated with the gene specific primers used for reverse transcription. They are designed to have similar hybridization properties and minimal cross reactivity Instead of cDNA

Prior to detection, the tags are amplified by T7 based in vitro transcription and/or an emulsion based PCR method. These methods result in unbiased amplification of tags. Thus, all genes are represented in correct proportion for gene expression study.