MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue.

نویسندگان

  • N A Datson
  • J van der Perk-de Jong
  • M P van den Berg
  • E R de Kloet
  • E Vreugdenhil
چکیده

Serial Analysis of Gene Expression (SAGE) is a powerful expression profiling method, allowing the analysis of the expression of thousands of transcripts simultaneously. A disadvantage of the method, however, is the relatively high amount of input RNA required. Consequently, SAGE cannot be used for the generation of expression profiles when RNA is limited, i.e. in small biological samples such as tissue biopsies or microdissected material. Here we describe a modification of SAGE, named microSAGE, which requires 500- to 5000-fold less starting material. Compared with SAGE, microSAGE is simplified due to incorporation of a 'single-tube' procedure for all steps from RNA isolation to tag release. Furthermore, a limited number of additional PCR cycles are performed. Using microSAGE gene expression profiles can be obtained from minute quantities of tissue such as a single hippocampal punch from a rat brain slice of 325 micrometers thickness, estimated to contain, at most, 10(5) cells. This method opens up a multitude of new possibilities for the application of SAGE, for example the characterization of expression profiles in tissue biopsies, tumor metastases or in other cases where tissue is scarce and the generation of region-specific expression profiles of complex heterogeneous tissues.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Serial analysis of gene expression in neurofibromatosis type 2-associated vestibular schwannoma.

HYPOTHESIS The genesis, morphology, and growth characteristics of vestibular schwannomas are determined by genetic alterations which vary gene transcript expression and this transcript expression can be qualitatively and quantitatively evaluated using the SAGE technique. By use of such technique, gene products with tumorigenic potential may be identified, providing insight and targets for futur...

متن کامل

Small amplified RNA-SAGE: an alternative approach to study transcriptome from limiting amount of mRNA.

Serial analysis of gene expression (SAGE) is a widely used and powerful technique to characterize and compare transcriptomes. Although several modifications have been proposed to the initial protocol with the aim of reducing the amount of starting material, unless additional PCR steps are added, the technique is still limited by the need for at least 1 micro g of total RNA. As extra PCR amplifi...

متن کامل

Improved NlaIII digestion of PAGE-purified 102 bp ditags by addition of a single purification step in both the SAGE and microSAGE protocols.

Despite the success of microarray technologies, serial analysis of gene expression (SAGE) still remains the only technique that allows an accurate quantitative and qualitative analysis of cell transcription in a variety of physiological and pathological conditions. Nevertheless, the efficiency of SAGE is limited by the numerous gel purification steps required and these increase the possibility ...

متن کامل

Semiquantitative RT-PCR analysis to assess the expression levels of iNOS in chicken macrophages

Abstract: iNOS is inducible by a variety of factors related to inflammation and referred to as inducible NOS(iNOS). It is regulated at the level of gene expression; once expressed, it produces NO at a high rate. iNOS gene-expression profiling is an important tool in understanding molecular markers of the responses of cells and tissues to external factors. In this article a semiquantitative reve...

متن کامل

Effect of Feeding Oak Acorn on Expression of IL-2, IL-13 and IFN-γ Genes in Bursa Fabricius Tissue of Broiler Chickens (Short Communication)

Today, the use of oak as a replacement feed in poultry diets is well documented. However, oak contains polyphenol compounds (tannins) as antinutrient factors resulted in limited usage in the poultry rations. Generally, consumption of feeds containing tannin could affect gene expression level of immune system. Therefore, the aim of this study was to investigate the effect of different levels of ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • Nucleic acids research

دوره 27 5  شماره 

صفحات  -

تاریخ انتشار 1999