A modified PCR-LongSAGE protocol identifies novel transcripts in human CD34⁺ bone marrow cells

¹ Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada
² Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; Genetics Program, University of British Columbia, Vancouver, British Columbia, Canada
³ Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver British Columbia, Canada
⁴ Genetics Program, University of British Columbia, Vancouver, British Columbia, Canada; Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver British Columbia, Canada
⁵ Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver British Columbia, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada
⁶ Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; Department of Medicine, and Experimental Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada

^* To whom correspondence should be addressed. E-mail: ceaves{at}bccrc.ca.

	Abstract

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 PCR (Q-RT-PCR) analyses and comparison of tag frequencies in replicate LongSAGE libraries produced from amplified and non-amplified 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 to be 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.