title: Quantitative assessment of gene expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain reaction.
abstract: OBJECTIVE
  Quantitative assessment of gene expression in stem cells is essential for understanding the molecular events underlying normal and malignant hematopoiesis. The aim of the present study was to develop a method for precise quantitation of gene expression in small subsets of highly purified CD34(+)CD38(-) stem cell populations.
  
  
  METHODS
  Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate housekeeping and drug resistance gene expression in cDNA obtained from 300 CD34(+)CD38(-) cells without cDNA amplification or nested PCR techniques.
  
  
  RESULTS
  Validation experiments in cell lines showed efficient, representative and reproducible gene amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in dilutional experiments and by detection of the low-copy gene PBGD. GAPDH was found to be a useful reference gene in normal and leukemic CD34(+)CD38(-) cells. In contrast, 18S rRNA content varied 100-fold to 1000-fold in these populations. Moreover, expression of 18S rRNA was significantly lower in leukemic CD34(+)CD38(+) cells compared to normal CD34(+)CD38(+) cells (p = 0.002). Expression of MDR-1 (18-fold, p < 0.0005), MRP-1 (3.8-fold, p < 0.05), and LRP (1.8-fold, NS) was higher in normal CD34(+)CD38(-) compared to CD34(+)CD38(+) cells.
  
  
  CONCLUSIONS
  Real-time quantitative RT-PCR is a valuable tool for precise quantitation of gene expression in small subsets of hematopoietic cells. Using this method, we showed the inappropriateness of 18S as a reference gene in these progenitors and the down-regulation of drug-resistance-related genes early in hematopoiesis.
authors.affiliation: Department of Hematology and Central Hematology Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands. H.raaijmakers@chl.azn.nl
authors.forename: Marc H G P
authors.initials: MH
authors.lastname: Raaijmakers
authors.forename: Liesbeth
authors.initials: L
authors.lastname: van Emst
authors.forename: Theo
authors.initials: T
authors.lastname: de Witte
authors.forename: Ewald
authors.initials: E
authors.lastname: Mensink
authors.forename: Reinier A P
authors.initials: RA
authors.lastname: Raymakers
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D000943:Antigens, Differentiation / Q000097:blood; D001854:Bone Marrow Cells / Q000166:cytology / Q000473:pathology / Q000502:physiology; D002460:Cell Line; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D019450:Genes, MDR; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D007938:Leukemia / Q000097:blood / Q000235:genetics / Q000473:pathology; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase / Q000097:blood; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012016:Reference Values; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D012337:RNA, Ribosomal, 18S; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1