影响RNA芯片探针杂交效率的因素分析

Factors Analysis of Oligodeoxyribonucleotide Probes for RNA Microarray

RNA芯片作为研究ncRNA的主要技术之一,已受到广泛关注。然而因RNA芯片中靶序列的单链特征,使得RNA芯片的探针设计比DNA复杂。本研究对加强型绿色荧光蛋白(EGFP)RNA序列进行覆瓦式探针设计,制作了RNA原位合成芯片,研究探针/靶序列杂交双链ΔG和Tm、靶序列二级结构以及探针的末端碱基对RNA芯片杂交信号强度的影响。结果表明,探针/靶序列杂交双链ΔG和Tm相同的探针间的杂交信号存在较大差异,探针/靶序列杂交双链ΔG和Tm与RNA芯片杂交信号之间未发现明显的规律性;靶序列二级结构的探针PT-sc参数与芯片杂交信号强度间呈较好的线性关系;探针5’末端碱基组成对芯片杂交信号强度也有影响,5’末端碱基为G/C的探针杂交信号总体上高于其邻近的5’末端碱基为A/T的探针的杂交信号,为RNA芯片的探针筛选提供一定的理论基础。

As one of main technologies on ncRNA research, the RNA microarray is receiving more at- tention, however, the probe design of RNA microarray is more complex than that of DNA microarray due to the single-chain characteristics of the target sequence. The RNA microfluidic picoarray for enhanced green fluorescent protein（EGFP） RNA transcript is designed using tiling probes in this study. The effects of free energy（△G） and melting temperature（Tin） of probe-target heteroduplex, secondary structure of tar- get and base in the end of probe are evaluated, thus lay the certain basis of the screening of the probe of RNA microarray. The results show that the same free energy（△G） and melting temperature（Tin） of probe- target heteroduplex have different hybridization signal intensity, there is no significant regularity between free energy（△G） and melting temperature（Tin） probe-target heteroduplex and hybridization signal intensity is observed, there is a linear relationship between PT-sc of probe which reflect the secondary structure in- formation of target and hybridization signal intensity. The base composition of the probe＇s 5＇-end also affects the hybridization signal intensity. The stronger hybridization signal intensity are usually obtained when the 5＇-end base of probes is G or C compared to the probes which 5＇-end base of probes is A or T.