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Figure 6 | Molecular Cancer

Figure 6

From: Genetic alteration and gene expression modulation during cancer progression

Figure 6

Serial analysis of gene expression (SAGE) library construction. This figure shows the steps in SAGE profiling. (A) An RNA population is reverse transcribed to cDNAs using oligo-T primers attached to magnetic beads. (B) cDNAs are collected and digested with the restriction endonuclease Nla III. (C) Linkers containing sequence recognized by BsmF I are ligated to the digested cDNAs. Sequence tags are released from the beads by BsmF I digestion (BsmF I cuts at a fixed distance downstream from its recognition site). (D) Released DNA tags are ligated together to form ditags. (E) Ditags are amplified and then digested with Nla III to remove the linkers. (F) Ditags are ligated together to form a concatemer which is then clones into a plasmid vector to generate a SAGE library. The identity and abundance of tags is deduced from DNA sequence analysis of plasmid clones of concatemated ditags. (G) Relative abundance of gene expression – between genes within the same RNA population or between samples – is deduced by counting sequence tags. Diagrams provided by Dr. K. Lonergan.

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