Microarray analysis using a limited amount of cells.

cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.

[Effect of growth factor on the phenotype of subpopulations and on the kinetics of CD34+ cells in the peripheral blood and in grafts of peripheral stem cells in patients with non-Hodgkin's lymphoma indicated for autologous peripheral blood stem cell transplantation]. / Vliv typu rustového faktoru na fenotyp subpopulací a kinetiku CD34+ bunek v periferní krvi a stepech periferních kmenových bunek pacientu s non-hodgkinskými lymfomy indikovaných k autologní transplantaci krvetvorby.

BACKGROUND: Peripheral blood stem cells are the preferred source for transplantation of hematopoiesis in patients with non-Hodgkin's lymphoma. Application of hematopoietic growth factors is a part of the mobilization chemotherapy regimen. Time overlap of the highest leukocyte and CD34+ cell count is required for optimal graft collection. Authors analyzed the effect of two growth factors (leridistim and filgrastim) on the kinetics and phenotype of CD34+ cells in patients with non-Hodgkin's lymphoma indicated for autologous peripheral blood stem cell transplantation. METHODS AND RESULTS: Authors analyzed phenotype of CD34+ cell subpopulations and their kinetics in peripheral blood and leukapheresis products by flow cytometry during mobilization and graft collection. Statistically significant differences in expression of lineage-committed antigens between growth factors were found (CD3, CD5--T-lineage, CD56 NK-lineage, CD20 for B-lineage, p < 0.05), as well as for lineage non-specific antigens (CD38, p < 0.05 and CD54, p < 0.01). The most significant divergence was observed between CD34+CD19+ subpopulations of leridistim and filgrastim stimulated blood and graft (p < 0.001). CONCLUSIONS: Expression of lineage-committed antigens on CD34+ subpopulations between two growth factors was statistically different. Kinetics of CD34+ cells during mobilization regimen with leridistim was not superior to filgrastim concerning the quality of graft.