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Blood, Vol. 107, Issue 4, 1582-1590, February 15, 2006
Coordination of intrinsic, extrinsic, and endoplasmic reticulum-mediated apoptosis by imatinib mesylate combined with arsenic trioxide in chronic myeloid leukemia
Blood Du et al.
107: 1582
Supplemental materials for: Du et al
Experimental Design
Cells were separately treated with 0.25 µM STI571, 1.0 µM ATO and the combination of the two, and harvested at 0, 3, 8, 12, 24, 48 and 72 hours of treatment, respectively. Un-treated K562 cells were collected at 0, 3, 8, 12, 24, 48 and 72 hours, and the collections were pooled, before used as the reference control. We applied cDNA microarray to analysis gene expression profiling of these 18 samples. Detailed information of microarray data (log(2) ratio of pixel numbers between red and green channels of each gene) is available in Table S1. RNA isolation and labeling
Harvested samples were then subjected to different assays and RNA preparations. Total RNA was prepared using TRIzol Reagent (Life Technologies, Gaithersburg, MD, USA), further purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantified using the RNA 6000 LabChip Kit on the 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) before subjected to reverse transcription labeling. 30 µg of each RNA sample was reversely transcribed into cDNA primed with oligo(dT) and labeled with either Cy3-dCTP or Cy5-dCTP using Superscript II (Life Technologies). The labeled probes were purified using Millipore PCR clean up columns (Millipore, Billerica, MA, USA), and concentrated to a final volume of 20 µl. Hybridization
Place the slides in a staining jar with Pre-treatment Solution (2×SSC/0.1%SDS/1%BSA) for 30 minutes at 50°C. Hybridization was then performed in the presence of 2 µg poly d(A), 3 × SSC, 0.1% SDS, 10 µg human Cot-1 DNA and equal amount of labeled target and reference cDNA at 65°C overnight. Washing was performed 4 × 5 min in 2×SSC, 0.1%SDS, 4 × 5 min in 0.2×SSC, 0.1%SDS, 4 × 5 min in 0.2×SSC, and a final wash of 1 minute in 0.02 × SSC. All washes were performed at 50°C. Fluorescent images of hybridized microarrays were obtained using a laser scanner Axon 4000B (Axon Instruments, Union City, CA, USA). Fluorescent intensities were quantified using GenePix Pro 4.1. Further normalization was used TIGR_MIDAS v 2.18 software. Hybridization for each sample was repeated at least twice and only data with high correlation coefficient (≥0.95) was further analyzed. Array design
Microarrays with 12,630 cDNA clones representing 10,647 genes, were fabricated in-house using a Generation III spotter (Amersham Biosciences). The cDNA clones were sequence verified and enriched with genes expressed in hematopoietic cells1,2. Among these cDNA clones include commercial clones from Invitrogene company. The Scorecard plate including positive control, negative control, dynamic range control, ratio control and housekeeping genes were spotted on per slide. Detailed information can be found at (http://www1.amershambiosciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=63004285&moduleid=165076#content.). Microarray slides were obtained from Fullmoon BioSystems (Sunnyvale, CA, USA). The clones were spotted in a final concentration of 200-400 fmol/µl in spotting buffer (50% DMSO) using 12 microspot pins to reach a complexity of 12,630 spots per slide. After spotting the slides were UV cross-linked (400 mJoules) and stored at room temperature. Reference List 1. Mao M, Fu G, Wu JS et al. Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning. Proc Natl Acad Sci U S A. 1998;95:8175-8180. 2. Zhang QH, Ye M, Wu XY et al. Cloning and functional analysis of cDNAs with open reading frames for 300 previously undefined genes expressed in CD34+ hematopoietic stem/progenitor cells. Genome Res. 2000;10:1546-1560.
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