Human bone-marrow mononuclear cells were obtained from the Poietics Company (Gaithersburg, MD).
Extracted molecule
total RNA
Extraction protocol
Human bone-marrow mononuclear cells were isolated from bone marrow with FicollyPaque solution and stored in liquid nitrogen. Cells from three donors were thawed at 37°C, pooled, and used immediately for the isolation of myeloid progenitor cells with CD15 magnetic beads (Dynal, Oslo, Norway). The cells isolated by CD15 magnetic beads were lysed directly with TRIzol reagent for isolation of total RNA. mRNA was purified from 5 mg of total RNA with oligo(dT)25 beads. cDNA was synthesized with a cDNA synthesis kit, and SAGE was performed according to the SAGE protocol with the exceptions described in the original paper. SAGE-tag sequences were collected with the Big-Dye sequencing kit and ABI377 sequencer, and tag sequences were extracted with SAGE 300 software.
Description
Lee S, Zhou G, Clark T, Chen J, Rowley JD, Wang SM. The pattern of gene expression in human CD15+ myeloid progenitor cells. Proc Natl Acad Sci U S A. 98(6):3340-5, 2001; Lee S, Chen J, Zhou G, Shi RZ, Bouffard GG, Kocherginsky M, Ge X, Sun M, Jayathilaka N, Kim YC, Emmanuel N, Bohlander SK, Minden M, Kline J, Ozer O, Larson RA, LeBeau MM, Green ED, Trent J, Karrison T, Liu PP, Wang SM, Rowley JD. Gene expression profiles in acute myeloid leukemia with common translocations using SAGE. Proc Natl Acad Sci U S A. 103(4):1030-5, 2006; We performed a genome-wide analysis of gene expression in primary human CD151 myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitativetinformation for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.
