title: A modified polymerase chain reaction-long serial analysis of gene expression protocol identifies novel transcripts in human CD34+ bone marrow cells.
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 (<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.
authors.affiliation: Terry Fox Laboratory, Vancouver, BC, Canada V5Z 1L3.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhao
authors.forename: Afshin
authors.initials: A
authors.lastname: Raouf
authors.forename: David
authors.initials: D
authors.lastname: Kent
authors.forename: Jaswinder
authors.initials: J
authors.lastname: Khattra
authors.forename: Allen
authors.initials: A
authors.lastname: Delaney
authors.forename: Angelique
authors.initials: A
authors.lastname: Schnerch
authors.forename: Jennifer
authors.initials: J
authors.lastname: Asano
authors.forename: Helen
authors.initials: H
authors.lastname: McDonald
authors.forename: Christina
authors.initials: C
authors.lastname: Chan
authors.forename: Steven
authors.initials: S
authors.lastname: Jones
authors.forename: Marco A
authors.initials: MA
authors.lastname: Marra
authors.forename: Connie J
authors.initials: CJ
authors.lastname: Eaves
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000378:metabolism*; D002469:Cell Separation; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D018952:Antigens, CD34; D018076:DNA, Complementary; D012333:RNA, Messenger