Identification and prevention of a GC content bias in SAGE libraries

Serial Analysis of Gene Expression (SAGE) is becoming a widely used gene expression profiling method for the study of development, cancer and other human diseases. Investigators using SAGE rely heavily on the quantitative aspect of this method for cataloging gene expression and comparing multiple SAGE libraries. We have developed additional computational and statistical tools to assess the quality and reproducibility of a SAGE library. Using these methods, a critical variable in the SAGE protocol was identified that has the potential to bias the Tag distribution relative to the GC content of the 10 bp SAGE Tag DNA sequence. We also detected this bias in a number of publicly available SAGE libraries. It is important to note that the GC content bias went undetected by quality control procedures in the current SAGE protocol and was only identified with the use of these statistical analyses on as few as 750 SAGE Tags. In addition to keeping any solution of free DiTags on ice, an analysis of the GC content should be performed before sequencing large numbers of SAGE Tags to be confident that SAGE libraries are free from experimental bias.     

High-performance liquid chromatography purification of 26-bp serial analysis of gene expression ditags results in higher yields,longer concatemers,and substantial time savings

In contrast to DNA chips, serial analysis of gene expression (SAGE) is not dependent on genes having been previously identified for their monitoring. Although useful, the method can be technically challenging, and particularly the last steps including concatenation and cloning may result in less than optimal results. We propose that many of the encountered problems can be attributed to the purification of the 26-bp ditags by polyacrylamide gel electrophoresis. Low yields, gel contaminants, potential exposure to degrading enzymes during handling and lengthy separation all disfavor the method. We introduce purification of 26-bp ditags by reverse-phase high-performance liquid chromatography (HPLC) using polystyrene/divinylbenzene columns and tetraethylammonium acetate buffer with acetonitrile as mobile phase. The method is fast and gives excellent results. Ditags purified by HPLC readily ligate to high-molecular-weight concatemers leading to their efficient cloning. The method should substantially facilitate the construction of SAGE libraries.     

Serial analysis of gene expression: rapid RT-PCR analysis of unknown SAGE tags.

In a pilot study on SAGE on Reed-Sternberg cells we have sequenced 1055 tags representing 701 genes. Screening of the GenBank database resulted in the identification of a corresponding gene or EST for 490 of them. For 211 of the tags no homology could be detected. A major problem of the serial analysis of gene expression (SAGE) approach is how to further analyse the unknown tags. We have developed an RT-PCR-based method, rapid analysis of unknown SAGE tags (RAST-PCR), to analyse the expression of the corresponding genes. This approach can be used as a screening method to investigate whether or not the gene is differentially expressed between several cell types of interest.     

基因表达的系列分析方法研究进展

基因表达的系列分析（SAGE）是探讨组织或器官在不同条件下基因表达丰度以及差异表达的一种有效方法。本文介绍了SAGE方法的详细机理并且对SAGE方法学的改进进行了综述。     

Serial analysis of gene expression (SAGE): unraveling the bioinformatics tools

Serial analysis of gene expression (SAGE) is a powerful technique that can be used for global analysis of gene expression. Its chief advantage over other methods is that it does not require prior knowledge of the genes of interest and provides qualitative and quantitative data of potentially every transcribed sequence in a particular cell or tissue type. This is a technique of expression profiling, which permits simultaneous, comparative and quantitative analysis of gene-specific, 9- to 13-basepair sequences. These short sequences, called SAGE tags, are linked together for efficient sequencing. The sequencing data are then analyzed to identify each gene expressed in the cell and the levels at which each gene is expressed. The main benefit of SAGE includes the digital output and the identification of novel genes. In this review, we present an outline of the method, various bioinformatics methods for data analysis and general applications of this important technology.     

Enhanced concatemer cloning-a modification to the SAGE (Serial Analysis of Gene Expression) technique.

The Serial Analysis of Gene Expression (SAGE) method, described in 1995 by Velculescu et al ., represents a powerful means to compare gene expression between two mRNA populations. An improvement to SAGE that removes contaminating linker molecules, which compromise the efficiency of the method, has been developed. This modification utilises biotinylated PCR primers, which generate biotinylated linkers at an early stage in the SAGE protocol, thus allowing removal of the unwanted linkers by binding to streptavidin-coated magnetic beads at a later stage. The application of this modification resulted in the rapid generation of high ditag yields and clones with large average insert sizes.