No-match ORESTES explored as tumor markers

Sequencing technologies together with new bioinformatics tools have led to the complete sequencing of various genomes. However, information regarding the human transcriptome and its annotation is yet to be completed. The Human Cancer Genome Project, using ORESTES (open reading frame EST sequences) methodology, contributed to this major objective by generating data from about 1.2 million expressed sequence tags (ESTs). Approximately 30% of these sequences did not align to ESTs in the public databases and were considered no-match ORESTES. On the basis that a set of these ESTs could represent new transcripts, we constructed a cDNA microarray. This platform was used to hybridize against 12 different normal or tumor tissues. We identified 3,421 transcribed regions not associated with annotated transcripts, representing 83.3% of the platform. The total number of differentially expressed sequences, with fold differences between tumor and normal samples of at least two, in one or more different tissues, was 1,007. Also, about 28% of analyzed sequences could represent non-coding RNAs (ncRNAs). Our data reinforces the knowledge of the human genome being pervasively transcribed, and point out molecular marker candidates for different cancers. To reinforce our data, we confirmed, by real-time PCR, the differential expression of 3 out of 8 potentially tumor markers in prostate tissues. A list of 1,007 differentially expressed sequences, as well as the 291 potentially non-coding molecular markers for different tumors was provided. Experiments were performed in duplicate, using dye-swap method. We used linearly amplified RNA samples (T7-based protocol), derived from 56 normal or tumor tissues from 12 distincts body localization, and a pool of RNAs obtained from 15 distinct human cell lines as reference. cDNA was synthesized in the presence of aminoallyl-dUTP (Sigma-Aldrich) and labeled using Alexa Fluor 555 or Alexa Fluor 647 dyes (Invitrogen).