Transcriptional analysis of select CfMNPV genes and some antisense transcripts by an oligonucleotide microarray

The temporal expression of the 23 CfMNPV genes representing all four temporal classes including its 7 unique genes were determined by a modified oligonucleotide-based two-channel DNA microarray. Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene was also detected by the array analysis. The expression of four host genes varied several fold throughout virus infection. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements. We first developed a novel normalization protocol using Cy5-labeled CfMNPV viral genomic DNA (vgDNA) as equimolar reference standards for each probe in order to overcome the inherent variability problem of the traditional microarray normalization procedures including use of internal standards. Cy3-labeled cDNA was from total RNA isolated at different times post infection of Cf203 insect cells infected with CfMNPV. Host genes were unsuitable for normalization between microarrays. The DNA microarray results were selectively validated by quantitative RT-PCR (qRT-PCR). The nature of the polyhedrin antisense transcription was further investigated using long range RT-PCR analysis. Keywords: Time course, detection of antisense transcripts, viral genomic DNA normalization Total RNA was isolated from Cf203 cells at 0, 3, 6, 12, 24 and 48 h post infection, as well as from mock-infected Cf203 cells. The Cy3-labeled cDNA derived from 20 μg total RNA was co-hybridized with 10 ng/μl vgDNA to each array at 42ºC. Seven hybridizations in each experiment, two independent hybridization experiments were performed, and therefore 14 samples were analyzed and included in the sample submission including the mock-infected sample.

本研究采用改良的基于寡核苷酸的双通道DNA微阵列技术，对涵盖全部4个时序类别的23个CfMNPV基因（包含其中7个特有基因）的时序表达模式进行了检测分析。此外，该微阵列分析还检测到部分选定的CfMNPV基因（包括多角体蛋白基因）的非编码（反义）链转录产物。在病毒感染过程中，4个宿主基因的表达量变化可达数倍。本次使用的微阵列芯片包含针对23个CfMNPV开放阅读框（Open Reading Frame, ORF）及其互补链的寡核苷酸探针。为克服传统微阵列归一化流程（包括内标使用）固有的实验变异性问题，本研究以Cy5标记的CfMNPV病毒基因组DNA（viral genomic DNA, vgDNA）作为每个探针的等摩尔参考标准，建立了一套全新的归一化流程。本研究中用于杂交的Cy3标记互补脱氧核糖核酸（complementary DNA, cDNA），来源于感染CfMNPV的Cf203昆虫细胞在感染后不同时间点提取的总RNA。宿主基因不适用于不同微阵列之间的归一化校正。本研究通过定量逆转录聚合酶链反应（quantitative Real-Time Polymerase Chain Reaction, qRT-PCR）对部分微阵列检测结果进行了选择性验证。本研究还采用长距离逆转录聚合酶链反应分析，进一步探究了多角体蛋白基因反义转录的特性。关键词：时序动态、反义转录本检测、病毒基因组DNA归一化。实验分别在感染后0、3、6、12、24、48小时的Cf203细胞以及未感染的假感染Cf203细胞中提取总RNA。将从20μg总RNA中逆转录得到的Cy3标记cDNA，与终浓度为10ng/μl的vgDNA共同杂交至每张微阵列芯片，杂交温度设置为42℃。本研究共开展2次独立杂交实验，每项实验包含7次杂交，总计分析14个样本（包含假感染样本）并将其纳入本次样本提交数据中。