科研之友
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摘要
It is well known that the gene expression profiling can be detected by RT-PCR singly, or which can be detected by cDNA, array in large numbers, however, to evaluate the expression of several targeted genes in a special regulation pathway, or some interested genes in a certain disease simultaneously, the methods were limited. So, development of a simple, robust, sensitive, specific and economic assay satisfied the need mentioned above was very useful, and a bead-based flow-cytometric multiplex assay was aimed to establish. Multiplex ligation-dependent probe amplification (MLPA) was employed to amplify several targeted cDNAs using only one pair identical primers, each MLPA probe consisted of two short synthetic oligonucleotides, and the tag which was coupled chemically to the fluorescent beads was complementary to one probe. Five beads with different fluorescence intensity coupled to RAC2, RhoBTB3, SPA-1, Rap1GAP and GAPDH were established. Biotinylated PCR amplicons were then hybridized to the complementary tag on each bead set. Bound amplicons were detected by flow cytometry using a streptavidin-linked reporter dye, PE. 111 BM specimens were analyzed in total, include RA(22), RAEB(22), RAEBt(9), AML(33), and control group (22, including hyperplastic anemia, iron deficiency anemia, aplastic anemia et al), and the difference of the transcriptional level of RAC2, RhoBTB3, SPA-1 and Rap I GAP relative to GAPDH were analyzed using wilcoxon non-parametric test and SNK method among different groups. The results were confirmed by RQ-PCR. The bead-based flow-cytometric array had an excellent sensitivity and a wide linear range, could get a positive signal for PCR product from 0.002 5 to 0.1 mu mol, the fine specificity was proved by no cross-hybridization signals presented among different bead set, and the reproducibility were also good enough(P < 0.001). The expression profiling of RAC2, RhoBTB3, SPA-1, Rap1GAP and GAPDH detected by this liquid bead-based flow-cytometric array were obtained, and there existed significant difference among 5 groups ( MDS-RA, -RAEB, -RAEBt, AML, and control group)for the relative expression to GAPDH of RAC2, RhoBTB3, SPA-1 and Rap1GAP (P < 0.0001, P = 0.049 1, P = 0.020 6 and P = 0.004 6 respectively). These results were validated by RQ-PCR, and the data obtained by each method had close linear correlation, the Pearson correlation coefficient was 0.930, 0.946, 0.945 and 0.921 for RAC2, RhoBTB3, SPA-1 and Rap1GAP respectively (P < 0.001 for all four). A bead-based flow-cytometric multiplex assay for rapid assessment of gene expression profiling has been built successfully, and it was validated by RQ-PCR.
- 出版日期2011-7
- 单位苏州大学
