Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34+ haemopoietic stem and progenitor cells.
Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high-fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon-gamma and tumour necrosis factor-alpha on normal primary CD34+ HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34+ cells to CD95-mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34+ HSC.
Attia
Mohamed A
MA
Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, London, UK.
Welsh
Jonathan P
JP
Laing
Kenneth
K
Butcher
Philip D
PD
Gibson
Frances M
FM
Rutherford
Tim R
TR
D018952:Antigens, CD34; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D005784:Gene Amplification; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016372:RNA, Antisense; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018952:Antigens, CD34; D001426:Bacterial Proteins; D016372:RNA, Antisense; D014409:Tumor Necrosis Factor-alpha; C074890:FliP protein, Bacillus