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First published online April 20, 2007
Journal of Experimental Biology 210, 1497-1506 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.000406

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Constructing the landscape of the mammalian transcriptome

Piero Carninci

Genome Science Laboratory, Discovery and Research Institute, RIKEN Wako Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan and Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

View larger version (37K): [in this window] [in a new window]   Fig. 1. Schematic representation of different methods for preparing full-length cDNA libraries. Starting from mRNA (top, in green with a polyA tail), first strand cDNA synthesis is associated with or followed by various options (A–D). (A) The resulting full-length cDNA/RNA hybrid (cDNA in yellow) is captured with a cap-binding protein; in the case of truncated cDNA/RNA hybrids the cap is removed by prior RNAse digestion. The cap-binding protein is immobilized on another support. (B) The cap-trapper protocol. A biotin molecule is added to the cap site; as in A, RNAse I removes the cap from the truncated cDNA/RNA hybrids, and the remaining full-length hybrids can be captured by streptavidin immobilized onto a support. After B, optionally, cDNA can be normalized and subtracted, otherwise after A or B it is denatured, subjected to second-strand cDNA synthesis and directly cloned. (C) The oligo-capping procedure, where an oligonucleotide is ligated to the mRNA instead of the cap structure. (D) The SMART oligonucleotide (a short, extended template at the 5' end of the RNA template; see text for details) is also copied into the cDNA. Priming the oligonucleotide at the 5' ends only allows for full-length cDNA selection. After C and D, PCR amplification is required before cloning and sequencing.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Transcriptome discovery does not reach a plateau. (A–D) Rate of gene discovery as a function of the accumulated number of sequences. Gene discovery is represented from the number of 3' end clusters identified. A and B refer to a mixed cell line library; C and D to an embryo pituitary subtracted library (Carninci et al., 2003). (A,C) Internal redundancy in terms of novelty of 3' ESTs (y axis) compared to the sequenced cDNAs (x axis) within the library. (B,D) Novelty rate of these two libraries versus the entire RIKEN database of 1.4 million 3' ESTs (axes as in A and B). Note that the discovery rate, although different, is far from reaching a plateau, suggesting the existence of a much larger number of yet undetected RNAs in each tissue/cell line. The pituitary gland library was prepared at the end of the project, and so far has identified more than 2000 new transcripts.