Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34⁺ haemopoietic stem and progenitor cells

Abstract

Summary. 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-c and tumour necrosis factor-a 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.

Toxicogenomics Applied to Hematotoxicology

“…In their reports [35], a large amount of supporting online materials and raw data including those for 4289 genes were attached, which were obtained by using Affymetrix systems (the above is excerpted with permission from NB Ivanova et al, Science 298: 601-604 (2002); copyright 2002 AAAS). Similar trials were performed by Park et al [37], Ma et al [38], and Attia et al [39]; however, the data obtained cannot be compared among the reports, because the microarray systems and fractionation methods used were different. Also, overlapping genes were rarely observed because the stem cell compartments sorted out were different.…”

Comparison of Two Probe Preparation Methods Using Long Oligonucleotide Microarrays

“…Similarly, Scheidl et al (20) and others (10,2123) have shown highly concordant results generated between amplified RNA and total RNA. In contrast, recent work has shown that gene expression profiling using amplified versus nonamplified RNA is not completely preserved and in some instances show poor correlations (15,24,25). Therefore, it is evident that there is a need for further detailed comparisons of data sets obtained from different labeling methodologies on identical platforms.…”

Immunomodulation by herpesvirus U51A chemokine receptor via CCL5 and FOG‐2 down‐regulation plus XCR1 and CCR7 mimicry in human leukocytes

“…To specifically identify targets of constitutive signaling, array analyses were performed on non‐induced cells using a restricted gene set microarray composed of gene targets relevant to infection and immunity. This array had over 380 genes represented as exon‐specific PCR products gridded on glass slides with a design based on minimal cross‐hybridization 17 (for a complete V2 gene list, see Materials and methods ). The U51A data were filtered for all genes with signal above mean + 3 SD of non‐homologous (negative) controls, then using a cut‐off of twofold or greater changes in gene expression with p values less than 0.05.…”