We have previously identified a panel of genes by gene expression profiling which differentially expressed between primary bone marrow CD34+ cell of MDS patients and the normal control cells. The expression pattern of these genes were also confirmed individually by qRT-PCR in expanded cohort of MDS patients. To further explore the feasibility of using proper number of these genes as biomarkers and evaluate the clinical significance in the diagnosis and prognostic prediction of MDS patients, a convenient, robust, sensitive, specific and less expensive method need to be developed. Therefore, endeavors have been made to establish a bead-based flow-cytometric multiplex assay.
To establish this method, a technique called Multiplex ligation-dependent probe amplification (MLPA) was employed to amplify several targeted cDNAs by using single pair of identical primers. Each MLPA probe consists of two short synthetic oligonucleotide, and the tag which was coupled chemically to the fluorescent beads was complementary to one probe. Just as a pilot study to simultaneously detect the expression of four genes of the Rho GTPase family, five beads with different fluorescence intensity coupled to RAC2, RhoBTB3, SPA-1, Rap1GAP and GAPDH were designed. Biotinylated PCR amplicons were then hybridized to the complementary tag on each bead set. Bound amplicons were then detected by flow cytometry using a streptavidin-linked reporter dye, PE. 111 BM specimens were analyzed in total, consisting of RA(22), RAEB(22), RAEBt(9), AML(33), and control group (22, including hyperplastic anemia, iron deficiency anemia, aplastic anemia etc). The difference in the transcriptional level of RAC2, RhoBTB3, SPA-1 and Rap1GAP relative to GAPDH were analyzed using wilcoxon non-parametric test and SNK method among different groups. The results were confirmed by qRT-PCR.
The bead-based flow-cytometric array had a excellent sensitivity and a wide linear range, could get a positive signal for PCR product from 0.0025 to 0.1umol, 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 thus obtained. A significant difference among five groups for the expression level of RAC2, RhoBTB3, SPA-1 and Rap1GAP relative to GAPDH was found (P<0.0001, P=0.0491, P=0.0206 and P=0.0046 respectively). For RAC2, no difference existed between AML and control groups, while both these two groups demonstrated significant difference compared with RA, RAEB and REABt groups. The RA group had the highest expression level, AML the lowest; and RAEB in between. For RhoBTB3, a significant difference could be seen between RAEBt and other groups, RAEBt had the highest level of RhoBTB3. There was no difference in SPA-1 expression level determined between each two groups. However, an increasing tendency from control group to RA, RAEB, and REABt could be observed, whereas the expression of SPA-1 dramatically dropped to the lowest level in AML. For Rap1GAP, differences were found between AML and other group; These results were validated by qRT-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).
Based on these preliminary results, A liquid bead-based flow-cytometric multiplex assay for a rapid assessment of gene expression profile had been successfully developed and validated by qRT-PCR. This method will facilitates the clinical usage of a certain group of Rho GTPase family genes as well as other appropriate genes as useful biomarkers in the diagnosis of MDS patients and provide interesting clues to further explore the pathogenesis of MDS.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.