Small amplified RNA-SAGE: an alternative approach to study transcriptome from limiting amount of mRNA
Small amplified RNA-SAGE: an alternative approach to study transcriptome from limiting amount of mRNA
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DOI：
10.1093/nar/gng024
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发表时间：
2003-03-15
影响因子：
14.9
通讯作者：
Vassart, G
中科院分区：
文献类型：
--
作者：
Vilain, C;Libert, F;Vassart, G
基因表达序列分析（SAGE）是一种广泛使用且功能强大的转录组表征和比较技术。尽管为了减少起始材料的数量，已经对最初的方案提出了一些修改，除非增加额外的PCR步骤，否则该技术仍然受到至少需要1杯总RNA的限制。由于额外的PCR扩增可能会引入代表性偏差，目前的SAGE方案并不完全适用于小的显微解剖组织样本的研究。我们在这里提出了一种替代方法，涉及包含SAGE标签的小mRNA片段的线性扩增。该程序允许从少至2500个细胞中制备超过100,000个标签的库。由5杯甲状腺总RNA制成的microSAGE文库与根据本方案由50 ng相同RNA制备的文库之间存在令人满意的相关性。
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 mug of total RNA. As extra PCR amplification might introduce representation biases, current SAGE protocols are not fully suitable for the study of small, microdissected tissue samples. We propose here an alternative method involving the linear amplification of small mRNA fragments containing the SAGE tags. The procedure allows preparation of libraries of over 100 000 tags from as few as 2500 cells. A satisfactory correlation was observed between a microSAGE library made from 5 mug of total thyroid RNA, and a library prepared from 50 ng of the same RNA preparation according to the present protocol.