Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis  

Abstract

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).

Robust analysis of 5′-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites

“…Using streptavidin magnetic beads, PCR amplicons are captured and then digested with NlaIII, a type II restriction enzyme that recognizes CATG and cuts DNA at every 250 bp. This enzyme has been widely used as a tagging enzyme to generate tags in many SAGE libraries 9,10 . After washing of the magnetic beads, the 5¢-NlaIII tags from two pools are mixed together and ligated to generate ditags using T 4 DNA ligase.…”

“…After washing of the magnetic beads, the 5¢-NlaIII tags from two pools are mixed together and ligated to generate ditags using T 4 DNA ligase. This ditag-based PCR amplification strategy is used because it provides a more faithful representation of the tags' expression frequencies in comparison to the use of individual or mono tags, which was previously tested in SAGE methodology 9,10 . The ligated NlaIII ditags are PCRamplified using primers specific to the 5¢-RNA oligo linkers.…”

“…The conventional SAGE methodology encounters many technical problems such as short inserts of concatamers and low cloning efficiency. To overcome these problems, we previously developed an improved LongSAGE protocol called robust-SAGE (RL-SAGE) 10,11 . Using this method, we have generated over a million RL-SAGE tags from plants (rice and maize) and fungus (Magnaporthe grisea) 12,13 .…”

Microarray analysis of gene expression during early development: a cautionary overview

“…Both of these methods are based on the presence of a poly(A) stretch on mRNAs. The SAGE method was designed to generate a single short tag (14-27 bp; Velculescu et al 1995, Gowda et al 2004) from every poly(A)-bearing RNA molecule, concatemerize them to produce a library of tags and systematically tally each transcript in a sample by sequencing the library. MPSS is a different approach where cDNA molecules are immobilized individually on a bead and duplicated to populate its surface.…”

Caracterização de perfis transcricionais de folhas e da região cambial de Eucalyptus grandis usando o SAGE

“…Inicialmente foi desenvolvido o SuperSAGE que se baseia no uso de uma enzima de restrição tipoIII para gerar tags maiores que 25 pb (MATSUMURA et al, 2003). Posteriormente, foi desenvolvido o LongSAGE, onde tags de 20pb são produzidas tanto a partir da região 5' quanto da região 3' dos transcritos, aumentando a especificidade de identificação de genes.Outras modificações foram também propostas para aumentar a eficiência da clonagem e tamanho dos insertos, reduzindo o custo das metodologias que produzem tags de maior tamanho (GOWDA et al, 2004). …”