A Modified Polymerase Chain Reaction-Long Serial Analysis of Gene Expression Protocol Identifies Novel Transcripts in Human CD34+ Bone Marrow Cells
Article first published online: 5 APR 2007
Copyright © 2007 AlphaMed Press
Volume 25, Issue 7, pages 1681–1689, July 2007
How to Cite
Zhao, Y., Raouf, A., Kent, D., Khattra, J., Delaney, A., Schnerch, A., Asano, J., McDonald, H., Chan, C., Jones, S., Marra, M. A. and Eaves, C. J. (2007), A Modified Polymerase Chain Reaction-Long Serial Analysis of Gene Expression Protocol Identifies Novel Transcripts in Human CD34+ Bone Marrow Cells. STEM CELLS, 25: 1681–1689. doi: 10.1634/stemcells.2006-0794
- Issue published online: 2 JAN 2009
- Article first published online: 5 APR 2007
- Manuscript Accepted: 26 MAR 2007
- Manuscript Received: 6 DEC 2006
- Long serial analysis of gene expression;
- Stem/progenitor cells;
- Bone marrow;
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 (<103 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.