低密度cDNA芯片技术的优化

Optimization of Low-density cDNA Microarray

为了建立稳定的低密度cDNA芯片技术平台,研究靶基因的最适长度、浓度、点样溶液种类及杂交反应动力学,并了解该芯片的重复性与可靠性.结果表明,杂交具有较好的特异性,不同长度(189～1078bp)、浓度(0.5g/L、1.0g/L、1.5g/L)的同一靶基因杂交信号强度无明显差别;以50%DMSO为点样溶液者杂交信号最好(P=0.0001).60℃杂交18h信号最佳(P<0.001).重复2次检测结果差异无显著性(P=0.348),重复性较好,其相关系数为0.588.与RT-PCR结果相比,相关系数为-0.778(P<0.0001),特异性为100%,灵敏度为80%(16/20),可靠性较好.

In order to construct a stable low density cDNA microarray technique, housekeeping genes were selected to study the most suitable target gene length, concentration, spotting solution and hybridizing dynamics. All target genes' mRNA expression in the same cells (SGC7901) were detected by the identical cDNA microarrays and by RT PCR for reproducibility and reliability. The hybridization signals had a good specificity. No signal showed in either negative control (HBV) or blank control (spotting solution only). There was neither significant difference in target gene lengths (189～1 078 bp) nor concentration difference ( 0.5 g/L, 1.0 g/L and 1.5 g/L). Among the three kinds of spotting solution (3×SSC, 50%DMSO and 0.5 mol/L carbonate buffer (pH=9.0)), 50%DMSO was the best (P=0.000 1). The longer of hybridizing time, the stronger of signal intensity, 18 hours was the best and signal intensity decreased after 24 hours (P<0.001). With respect to the hybridizing temperature, 60 ℃was the strongest (P<0.001). The signal difference was not significant in two repeated examination (t= 0.941,P=0.348) , the correlation coefficient was 0.588. In comparison with RT PCR, the correlation coefficient was 0.778 (P<0.000 1) , specificity was 100%and sensitivity was 80%. The different target gene length between 200 bp and 1000 bp had got the same hybridization signals. 50%DMSO spotting solution and the target gene concentration at 0.5 g/L were suitable for good hybridization. To hybridize 18 hours at 60 ℃was the best condition of the low density cDNA microarray. The reproducibility and reliability was good. The specificity was superior to RT PCR and sensitivity inferior to it.