Quantitative analysis of CD34+ stem cells using RT-PCR on whole cells.
We have employed RT-PCR of whole cells to develop a quantitative method for estimating the number of rare cells expressing a unique mRNA in a large, mixed population of cells. We have demonstrated that RT-PCR can be done on whole cells without the need for extraction of the RNA. This allows for a great saving of time and effort, as well as allowing quantitative analysis to be based on the total number of cells analyzed in a given aliquot and the presence or absence of the specific RT-PCR product. We have employed a limiting dilution series on whole cells, with multiple aliquots at each cell concentration to achieve more statistical power in the analysis of a rare cell type. We have used a nested amplification of the CD34 mRNA to be able to detect a single cell expressing the CD34 mRNA in a larger population of non-CD34-expressing cells. We demonstrate that by using this technique, cells from blood and bone marrow containing the CD34 mRNA can be followed quantitatively during a multistep purification involving immunoadsorption followed by fluorescence-activated cell sorting. We also demonstrate that many cells that express the CD34 protein on their surface no longer contain detectable levels of CD34 mRNA, a phenomenon that appears to be developmentally regulated.
Molesh
D A
DA
CellPro, Inc., Bothell, Washington 98021.
Hall
J M
JM
D015703:Antigens, CD / Q000235:genetics*; D018952:Antigens, CD34; D001483:Base Sequence; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D013223:Statistics as Topic
D015703:Antigens, CD; D018952:Antigens, CD34; D012333:RNA, Messenger