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This Article Free via Open Access: OA OA Full Text Full Text (PDF) Supplemental Data OA All Versions of this Article: 2006-0794v1 25/7/1681    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zhao, Y. Articles by Eaves, C. J. Search for Related Content PubMed PubMed Citation Articles by Zhao, Y. Articles by Eaves, C. J.

STEM CELL GENETICS AND GENOMICS

A Modified Polymerase Chain Reaction-Long Serial Analysis of Gene Expression Protocol Identifies Novel Transcripts in Human CD34⁺ Bone Marrow Cells

^aTerry Fox Laboratory and
^cMichael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada;
^bGenetics Program and the
Departments of ^dMedical Genetics,
^eMedicine, and
^fExperimental Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada

Key Words. Long serial analysis of gene expression • Stem/progenitor cells • Bone marrow • Transcriptome

Correspondence: Connie. J. Eaves, Ph.D., Terry Fox Laboratory, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3. Telephone: 604-675-8122; Fax: 604-877-0712; e-mail: ceaves{at}bccrc.ca

Received December 6, 2006; accepted for publication March 26, 2007.
First published online in STEM CELLS EXPRESS   April 5, 2007.



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.