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Supplemental Material, sj-pdf-1-ang-10.1177_00033197221082712 for Long-Term Blood Pressure Exposure From Childhood and Early Vascular Aging in Midlife: A 30-Year Prospective Cohort Study by Yu Yan, Yu Cao, Qiong Ma, Keke Wang, Yueyuan Liao, Yue Sun, Chen Chen, Jiawen Hu, Wenling Zheng, Chao Chu, Yang Wang, and Jianjun Mu in Angiology

A complementary DNA (cDNA) fragment library from SH-SY5Y cells is constructed using a restriction display polymerase chain reaction (RD-PCR) technique. Messenger RNA (mRNA) is extracted from SH-SY5Y cells and single-strand cDNA synthesised using an anchored oligo primer (dT18). The second strand is produced by nick translation. The double strands are cleaved with the restriction enzyme Sau3AI and the fragments ligated with universal linker. The products are amplified with universal primers and selected primers, ligated into the pMDI8-T vector, and then sequenced. The library constructed contained 136 subgroups, each comprising seven to 12 cDNA fragments. RD-PCR proved a simple, effective way to construct a cDNA library, and this will contribute to the investigation of gene expression in the neuron in future microarray studies.

The oncoprotein E6 of high-risk human papillomavirus (HPV) types promotes cell proliferation and contributes to carcinogenesis of HPV-positive cervical cancer cells. In this study, we used small interfering RNA (siRNA) technology to silence the E6 gene in HPV-18-transformed human cervical cell line HeLa and determined the effects of E6 gene knockdown on the cell by using microarray-based gene expression profiling coupled with gene functional classification with bioinformatics methods. Silencing RNA prepared by siRNA expression cassettes against HPV-18 E6 gene could significantly inhibit E6 gene expression and induce HeLa cells to apoptosis. The microarray analysis identified 359 differentially expressed genes containing 307 up-regulated and 52 down-regulated genes. We analyzed the gene functions and cellular pathways in detail, including cell cycle-related genes, CCNG1 and p21; apoptosis-related genes, CASP4, CASP6, IGFBP3, and DFFA; ubiquitin proteolysis pathway-related genes, UBE3A and UBE2C; keratinocyte differentiation-related genes, KRT4, KRT6E, and KRT18; and antioncogenes, RECK and VEL. In addition, it can be concluded that cellular apoptosis induced by HPV-18 E6 siRNA mainly depends on the P53 and ubiquitin proteolysis pathway to regulate gene expression, consequently inhibiting cell proliferation and promoting cell apoptosis. Meanwhile, activation of antioncogene and upper regulation of immunization-related genes signified the degression of the malignant extent of tumor cells after E6 inhibition. Our approach, which combines the use of siRNA-mediated gene silencing, microarray screening, and functional classification of differential genes, can be used in functional genomics study to elucidate the role of E6 oncogene in the carcinogenesis of HPV-18 and provide some possible targets for clinical treatment and drug development of cervical cancer.

Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility of using approximately 70mer oligonucleotide microarray for detection and genotyping of HPV. The approximately 70mer type-specific oligonucleotide probes of four different types HPV were designed by using biological software Array designer 2.0, which analyzed the whole genome sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls. These results demonstrated that approximately 70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for the discriminate diagnosis of multiple pathogens.

A new molecular-baiting method was studied by retrieving targeted gene fragments from an oligonucleotide microarray bait after hybridization. To make the microarray bait, 70-mer oligonucleotides that were designed to specifically represent the SSA1 gene of Saccharomyces cerevisiae were printed on the slide. Samples of the Saccharomyces cerevisiae mRNA were extracted and labeled by the RD-PCR (Restriction Display PCR) method using the Cy5-labelled universal primer, then applied for hybridization. The sample fragments that hybridized to the microarray were stripped, and the eluted cDNAs were retrieved and cloned into the pMD 18-T vector for transformation, plasmid preparation, and sequencing. BLAST searching of the GenBank database identified the retrieved fragments as being identical to the SSA1 gene (from 2057-2541bp). A new method is being established that can retrieve the sample fragments using an oligo-microarray-bait.

Vaccinia virus is a member of the orthopoxvirus group, to which also belongs variola virus, one of the most hazardous pathogens known to man. To establish a model system to detect orthopoxviruses, a vaccinia oligonucleotide microarray is designed, produced and tested. Vaccinia virus is used to test the prepared microarrays. The virus DNA samples in different propagation phases are extracted and hybridised with the oligonucleotide microarray. The results showed that the oligonucleotide microarray can detect vaccinia virus with high specificity and sensitivity.

This study explores the restriction display-polymerase chain reaction (RD-PCR) application of a new chip-based nucleic acid analysis system (Agilent 2100 bioanalyser) in a gene differential expression study. Total RNAs is extracted from Saccharomyces cerevisiae, double-stranded complementary DNA (cDNA) is synthesised by reverse transcription from the purified messenger RNA (mRNA), RD-PCR conducted to obtain the cDNA fragments and bioanalyser and agarose gel electrophoresis compared for the analysis of RD-PCR products. The bioanalyser proved to be faster and more sensitive in separating and detecting gene fragments, and was also able to compare different gene fragments quantitatively. Using this technology, comparison of several differential gene fragments is performed.

(2002). Re-use of a stripped cDNA microarray. British Journal of Biomedical Science: Vol. 59, No. 2, pp. 112-113.