Vilain Catheline, Vassart Gilbert
Institute of Interdisciplinary Research (IRIBHM), Univeristé Libre de Bruxelles, Brussels, Belgium.
Methods Mol Biol. 2004;258:135-52. doi: 10.1385/1-59259-751-3:135.
Serial analysis of gene expression (SAGE) is a powerful genome-wide analytic tool to determine expression profiles. Since its description in 1995 by Victor Velculescu et al., SAGE has been widely used. Recently, the efficiency of the method has been emphasized as a means to identify novel transcripts or genes that are difficult to identify by conventional methods. SAGE is based on the principle that a 10-base pair (bp) cDNA fragment contains sufficient information to unambiguously identify a transcript, provided it is isolated from a defined position within this transcript. Concatenation of these sequence tags allows serial analysis of transcripts by sequencing multiple tags within a single clone. Extraction of sequence data by computer programs provides a list of sequence tags that reflect both qualitatively and quantitatively the gene expression profile. Several modifications to the initial protocol allowed to start from 1 microg total RNA (or 10(5) cells). In order to reduce the amount of input RNA, protocols including extra polymerase chain reaction (PCR) steps were designed. Linear amplification of the mRNA targets might have advantage over PCR by minimizing biases introduced by the amplification step; therefore we devised a SAGE protocol in which a loop of linear amplification of RNA has been included. Our approach, named "small amplified RNA-SAGE" (SAR-SAGE) included a T7 RNA polymerase promoter within an adapter derived from the standard SAGE linker. This allowed transcription of cDNA segments, extending from the last NlaIII site of transcripts to the polyA tail; these small amplified RNAs then serve as template in a classical (micro)SAGE procedure. As the cDNAs are immobilized on oligo(dT) magnetic beads, several rounds of transcription can be performed in succession with the same cDNA preparation, with the potential to increase further the yield in a linear way. Except for the transcription step itself, the present procedure does not introduce any extra enzymatic reaction in the classical SAGE protocol, it is expected to keep the representation biases associated with amplification as low as possible.
基因表达序列分析（SAGE）是一种强大的全基因组分析工具，用于确定表达谱。自1995年Victor Velculescu等人描述以来，SAGE已被广泛使用。最近，该方法的效率作为一种识别传统方法难以鉴定的新转录本或基因的手段而受到重视。SAGE基于这样一个原理：一个10个碱基对（bp）的cDNA片段包含足够的信息来明确鉴定一个转录本，前提是它是从该转录本内的一个确定位置分离出来的。这些序列标签的串联允许通过对单个克隆内的多个标签进行测序来对转录本进行序列分析。通过计算机程序提取序列数据可提供一个序列标签列表，该列表在定性和定量上都反映了基因表达谱。对初始方案的几次修改使得可以从1微克总RNA（或10⁵个细胞）开始。为了减少输入RNA的量，设计了包括额外聚合酶链反应（PCR）步骤的方案。mRNA靶标的线性扩增可能比PCR更具优势，因为它能将扩增步骤引入的偏差降至最低；因此，我们设计了一种SAGE方案，其中包含了RNA线性扩增环。我们的方法名为“小扩增RNA-SAGE”（SAR-SAGE），在源自标准SAGE接头的接头内包含一个T7 RNA聚合酶启动子。这允许转录cDNA片段，从转录本的最后一个NlaIII位点延伸到聚腺苷酸尾；这些小扩增RNA随后在经典（微量）SAGE程序中用作模板。由于cDNA固定在寡聚（dT）磁珠上，同一cDNA制备物可以连续进行几轮转录，有可能以线性方式进一步提高产量。除了转录步骤本身外，本程序在经典SAGE方案中不引入任何额外的酶促反应，预计能将与扩增相关的代表性偏差保持在尽可能低的水平。
