Zhao Yun, Raouf Afshin, Kent David, Khattra Jaswinder, Delaney Allen, Schnerch Angelique, Asano Jennifer, McDonald Helen, Chan Christina, Jones Steven, Marra Marco A, Eaves Connie J
Terry Fox Laboratory, Vancouver, BC, Canada V5Z 1L3.
Stem Cells. 2007 Jul;25(7):1681-9. doi: 10.1634/stemcells.2006-0794. Epub 2007 Apr 5.
Transcriptome profiling offers a powerful approach to investigating developmental processes. Long serial analysis of gene expression (LongSAGE) is particularly attractive for this purpose because of its inherent quantitative features and independence of both hybridization variables and prior knowledge of transcript identity. Here, we describe the validation and initial application of a modified protocol for amplifying cDNA preparations from <10 ng of RNA (<10(3) cells) to allow representative LongSAGE libraries to be constructed from rare stem cell-enriched populations. Quantitative real-time polymerase chain reaction (Q-RT-PCR) analyses and comparison of tag frequencies in replicate LongSAGE libraries produced from amplified and nonamplified cDNA preparations demonstrated preservation of the relative levels of different transcripts originally present at widely varying levels. This PCR-LongSAGE protocol was then used to obtain a 200,000-tag library from the CD34+ subset of normal adult human bone marrow cells. Analysis of this library revealed many anticipated transcripts, as well as transcripts not previously known to be present in CD34+ hematopoietic cells. The latter included numerous novel tags that mapped to unique and conserved sites in the human genome but not previously identified as transcribed elements in human cells. Q-RT-PCR was used to demonstrate that 10 of these novel tags were expressed in cDNA pools and present in extracts of other sources of normal human CD34+ hematopoietic cells. These findings illustrate the power of LongSAGE to identify new transcripts in stem cell-enriched populations and indicate the potential of this approach to be extended to other sources of rare cells. Disclosure of potential conflicts of interest is found at the end of this article.
转录组分析为研究发育过程提供了一种强大的方法。基因表达的长序列分析（LongSAGE）因其固有的定量特性以及不受杂交变量和转录本身份先验知识的影响，在这一目的上特别具有吸引力。在此，我们描述了一种改良方案的验证和初步应用，该方案用于从<10 ng的RNA（<10³个细胞）扩增cDNA制备物，以便从富含干细胞的稀有群体构建具有代表性的LongSAGE文库。通过定量实时聚合酶链反应（Q-RT-PCR）分析以及对由扩增和未扩增的cDNA制备物产生的重复LongSAGE文库中标签频率的比较，证明了最初以广泛不同水平存在的不同转录本的相对水平得以保留。然后使用这种PCR-LongSAGE方案从正常成人骨髓细胞的CD34⁺亚群中获得了一个包含200,000个标签的文库。对该文库的分析揭示了许多预期的转录本，以及先前未知存在于CD34⁺造血细胞中的转录本。后者包括许多新标签，这些标签映射到人类基因组中的独特且保守的位点，但先前未被鉴定为人细胞中的转录元件。Q-RT-PCR用于证明这些新标签中的10个在cDNA文库中表达，并存在于其他来源的正常人类CD34⁺造血细胞提取物中。这些发现说明了LongSAGE在鉴定富含干细胞群体中的新转录本方面的能力，并表明这种方法有可能扩展到其他稀有细胞来源中。潜在利益冲突的披露见本文末尾。
