Gowda Malali, Jantasuriyarat Chatchawan, Dean Ralph A, Wang Guo-Liang
Department of Plant Pathology, Ohio State University, Columbus, Ohio 43210, USA.
Plant Physiol. 2004 Mar;134(3):890-7. doi: 10.1104/pp.103.034496.
Serial analysis of gene expression (SAGE) is a widely used technique for large-scale transcriptome analysis in mammalian systems. Recently, a modified version called LongSAGE (S. Saha, A.B. Sparks, C. Rago, V. Akmaev, C.J. Wang, B. Vogelstein, K.W. Kinzler [2002] Nat Biotechnol 20: 508-512) was reported by increasing tag length up to 21 bp. Although the procedures for these two methods are similar, a detailed protocol for LongSAGE library construction has not been reported yet, and several technical difficulties associated with concatemer cloning and purification have not been solved. In this study, we report a substantially improved LongSAGE method called Robust-LongSAGE, which has four major improvements when compared with the previously reported protocols. First, a small amount of mRNA (50 ng) was enough for a library construction. Second, enhancement of cDNA adapter and ditag formation was achieved through an extended ligation period (overnight). Third, only 20 ditag polymerase chain reactions were needed to obtain a complete library (up to 90% reduction compared with the original protocols). Fourth, concatemers were partially digested with NlaIII before cloning into vector (pZEro-1), greatly improving cloning efficiency. The significant contribution of Robust-LongSAGE is that it solved the major technical difficulties, such as low cloning efficiency and small insert sizes associated with existing SAGE and LongSAGE protocols. Using this protocol, one can generate two to three libraries, each containing over 4.5 million tags, within a month. We recently have constructed five libraries from rice (Oryza sativa), one from maize (Zea mays), and one from the rice blast fungus (Magnaporthe grisea).
基因表达系列分析（SAGE）是一种在哺乳动物系统中广泛用于大规模转录组分析的技术。最近，一种名为LongSAGE的改进版本（S. Saha、A.B. Sparks、C. Rago、V. Akmaev、C.J. Wang、B. Vogelstein、K.W. Kinzler [2002]《自然生物技术》20: 508 - 512）被报道，其标签长度增加到了21 bp。尽管这两种方法的步骤相似，但尚未有关于LongSAGE文库构建的详细方案报道，并且与串联体克隆和纯化相关的几个技术难题也尚未解决。在本研究中，我们报道了一种名为稳健型LongSAGE（Robust-LongSAGE）的显著改进的LongSAGE方法，与先前报道的方案相比，它有四个主要改进。首先，少量的mRNA（50 ng）就足以构建文库。其次，通过延长连接时间（过夜）实现了cDNA接头和双标签的形成增强。第三，仅需20次双标签聚合酶链反应就能获得完整的文库（与原始方案相比减少了多达90%）。第四，在克隆到载体（pZEro-1）之前，用NlaIII对串联体进行部分消化，大大提高了克隆效率。稳健型LongSAGE的重要贡献在于它解决了诸如与现有SAGE和LongSAGE方案相关的克隆效率低和插入片段小等主要技术难题。使用该方案，一个人可以在一个月内生成两到三个文库，每个文库包含超过450万个标签。我们最近已经从水稻（Oryza sativa）构建了五个文库，从玉米（Zea mays）构建了一个文库，从稻瘟病菌（Magnaporthe grisea）构建了一个文库。
