高性价比寡核苷酸基因表达谱芯片的制备方法

Fabrication of cost-effective expression profiling oligonucleotide microarrays

目的探讨探针长度对寡核苷酸(Oligo)基因芯片杂交信号的影响。方法以大肠杆菌基因表达信息作为实验模型,根据已有大肠杆菌全基因组芯片数据选择覆盖高、中、低杂交信号强度的20个大肠杆菌基因,针对该20个基因分别设计59-mer和70-mer长度Oligo探针,并将2种探针点制在同一张芯片中,同时设阳性对照探针和阴性对照点样液。提取大肠杆菌RNA,经过反转录、扩增、荧光标记后与芯片进行杂交反应,采用激光共聚焦扫描仪扫描芯片,利用数据分析软件提取探针的杂交信号值并进行显著性分析。结果大肠杆菌28SrRNA和18SrRNA条带清晰,无降解带出现,质量合格。芯片杂交结果显示59-mer和70-mer长度探针的杂交效率和杂交信号差异无统计学意义(P=0.9810),阳性对照探针出现阳性杂交信号,阴性对照点样液未检测到杂交信号,符合质控要求。结论59-mer长度探针可用来制备Oligo基因芯片,这不仅降低了基因芯片制作成本,而且将推动基因芯片技术更为普及的应用。

Objective To study the effect of oligonucleotide probe length on hybridization signals in order to fabricate cost-effective expression profiling microarrays. Methods The effect of oligonucleotide probe length on hybridization signals was studied through expression profiling of E.coli. Twenty E.coli genes,which represent low,median and high expression level respectively were chosen from the E.coli microarray database.Oligo probes of 59-mer and 70-mer,the former being more cost-effective than the latter,were designed and printed on a same slide.Positive control and spotting buffer as negative control were also spotted on the slide.Fluorescence labeled DNA were prepared from total RNA of E.coli through reverse transcription and RNA amplification,then hybridized with the array.After hybridization,slides were scanned with a confocal scanner and hybridization signals were extracted from images. Results The quality of total RNA was assessed,showing the 28S rRNA and 18S rRNA was intact.Our results indicated that there were no significant differences between the 59-mer probes and 70-mer probes in the hybridization efficiency and signal intensity（P= 0.9810）.In addition,the positive probes had strong intensity signal,while the negative spots had no signal as expected. Conclusions The cost-effective 59-mer oligo probes could be used to fabricate the expression profling microarray while retain a comparable result with longer probes.This can help to promote the extensive application of DNA microarray technology.