CD34 Cell Selection and Nucleic Acid Preparation

Fresh bone marrow is processed immediately after aspiration to select the CD34+ cells within the next 2 hr. CD34+ cells are purified by high-gradient magnetic cell separation (MACS) using superparamagnetic streptavidin microparticles. Yield and purity of the positively selected CD34+ cells are evaluated by flow cytometry.

Total RNA is extracted using TRIzol (Life Technologies, Grand Island, NY) according to the manufacturer's protocol with minor modifications. In addition, RNA is purified by the RNeasy system (Qiagen, Inc., Valencia, CA) following the manufacturer's advice. The quality of RNA is critical for any microarray experiment. To assure that the gene expression measured by microarray assay is not affected by degradation of the RNA extracted from the purified CD34+ cells, the Bioanalyzer system (Agilent, Waldbronn, Germany) is used to evaluate the quality of the RNA. Figure 1 shows the Bioanalyzer pattern of a high-quality RNA compared with a pattern that is related to degradation of RNA.

Fig. 1 Quality of RNA from CD34+ cells as determined by the Bioanalyzer system. (a) Analysis image of a high-quality total RNA extracted from CD34+ cells. Beside the marker spike (left), the 18S and 28S spikes (right) are clearly separated. There is no significant degradation of RNA. (b) Image of a degraded total RNA. Beside the marker spike, there is no clear separation of the 18S and/or 28S fraction demonstrating the high level of RNA degradation. (View this art in color at www.dekker.com.)

Fig. 1 Quality of RNA from CD34+ cells as determined by the Bioanalyzer system. (a) Analysis image of a high-quality total RNA extracted from CD34+ cells. Beside the marker spike (left), the 18S and 28S spikes (right) are clearly separated. There is no significant degradation of RNA. (b) Image of a degraded total RNA. Beside the marker spike, there is no clear separation of the 18S and/or 28S fraction demonstrating the high level of RNA degradation. (View this art in color at www.dekker.com.)

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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