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TSINGHUA UNIVERSITY Research Experience Beijing 100084 P.R.CHINA Rongrong Zhou Research Experience GRADUATE RESEARCH: Jul., 2004 – present: Graduate student, Department of Biological Science and Biotechnology (DBSB), Tsinghua University (THU); Microarray & Bioinformatics Department in National Engineering Research Center for Beijing Biochip Technology (NERCBBT) Master’s thesis: Detection mRNA of placental origin in maternal plasma by cDNA microarray Supervisor: Liang Zhang and Yuxiang Zhou Backgroud: Noninvasive prenatal diagnosis is a long-sought goal in human genetics. Recent interest in cell-free DNA in plasma and serum has led to the discovery of fetal DNA in maternal plasma. This noninvasive source of fetal nucleic acid has already been shown to be clinically valuable in the prenatal investigation of many conditions. Moreover, a number of investigators have shown that in addition to DNA, RNA is also present in the plasma of human subjects, particularly those with cancer. Direct evidences have shown that the placenta is an important source of fetal nucleic acid release into maternal plasma by demonstrating that mRNA transcripts from two placenta-expressed genes, namely the genes coding for human placental lactogen (hPL) and the subunit of human chorionic gonadotropin (hCG), are readily detectable in maternal plasma. Limited by the throughput of real-time quantitative RT-PCR which is currently used in this field, it’s difficult to detect many mRNA transcripts of placenta-expressed genes simultaneously. However, the advent of microarray technology will allow the detection of a great deal of these RNA and the very rapid development of new plasma RNA markers, which can be used for prenatal monitoring. Methods: In this project, we separate the plasma from maternal blood by centrifuge and pass it through filter with pore sizes of 5m. Then LS TRIzol was added to extract RNA. In the following process, RNA was amplificated by two-step cRNA synthesis and labeled with Cy5 or Cy3 by Klenow enzyme after reverse transcription. Finally, the labeled targets were hybridized with our cDNA chips and the arrays were scanned with a ScanArray Express scanner. Sep., 2003 – Jun., 2004: Graduate student, DBSB, THU; Microarray & Bioinformatics Department in National Engineering Research Center for Beijing Biochip Technology (NERCBBT) Master’s thesis: Genome-wide oligonucleotide microarray analysis of gene-expression profiles in placentas of preeclamptic pregnancies 1 TSINGHUA UNIVERSITY Research Experience Beijing 100084 P.R.CHINA Rongrong Zhou Supervisor: Liang Zhang and Yuxiang Zhou Backgroud: Preeclampsia is a multisystem disorder specific to human pregnancy which is characterized by gestational hypertension, fluid retention, and proteinuria. The hypertensive disorders of pregnancy affect up to 8% of all gestations. Although preeclampsia has been recognized as a pregnancy disorder since antiquity, the pathogenesis is not understood despite decades of research and the syndrome of preeclampsia is still one of the major pregnancy disorders and the leading cause of pregnancy-related maternal and fetal morbidity and mortality. Microarray analysis is a technology that allows the characterization of a whole-genome expression by showing the relative transcript levels of thousand of genes in one experiment, thereby speeding the identification of differential expression profiles. At present, large-scale gene expression studies based on microarray analysis have been applied to observe the gene expression patterns in human placentas from pregnancies complicated by preeclampsia in the third trimester. However, a whole genome-scale gene expression study has not been applied up to now to observe the gene expression patterns in human placentas from pregnancies complicated by preeclampsia. Clearly, further insight is required in this area given that the overall result of the candidate gene studies are still rarely in accordance with each other. Methods: Placentas tissues from five normal pregnancies and five pregnancies complicated by preeclampsia were collected. mRNA level of five preeclamptic placentas were examined respectively using a genome-wide 70-mer oligonucleotide microarray (21,329 human genes) in comparison with the pooled control consisting of total RNA from five normotensive placentas after the control cDNA and experimental cDNA were labeled by Cy5 and Cy3 respectively. Results: Over one hundred genes were found consistently down or up-regulated in at least four experiments. Many genes related to unbalance of reactive oxygen metabolites in placenta, abnormal trophoblast invasion, disorders of lipoprotein metabolism and signal transduction were among the differentially expressed genes, some of which had been reported to have close correlation to the pathology of preeclampsia. Most results of microarray analysis were also confirmed by real-time PCR. Jan., 2004-Apr., 2004 Graduate student, DBSB, THU; NERCBBT Research of biostatistics and bioinformatics Supervisor: Liang Zhang and Yuxiang Zhou Still with its high-throughput, microarray brings out thousands and ten thousands data. How to manage and analyze so many large and complex data effectively is a great challenge to bioinformatics. It is inevitable to have random error from various origins in the process of microarray. How to distinguish and remove these errors is the prerequisite to get the correct 2 TSINGHUA UNIVERSITY Research Experience Beijing 100084 P.R.CHINA Rongrong Zhou result. We analyzed the errors from available microarray data and then put forward a new kind of error model and a robust algorithm to estimate the parameters of the model. Applying to real and simulated data, this model and algorithm were proved to be correct and efficient. Lastly, combining this error model with Z-testing, a full statistical model was set up for data analysis, to select the differentially expressed genes from the result of microarray. Except for random error, there is also systematic error in the experiment of microarray. We can get the correct fold change of gene expression by normalization to eliminate this systematic error, between samples. We compared commonly used normalization methods and then developed an improved and robust algorithm, which we called malm, based on linear regression analysis. It was proved that malm could be effectively applied to real and stimulated data. Especially, based on its stability, malm can also be used to normalize biased expression data of microarray. Sep., 2002 – Jul., 2003: Graduate student, DBSB, THU; HLA typing Department in NERCBBT Supervisor: Huafang Gao and Yuxiang Zhou During this period, I participated in the project of HLA typing chip, the principle of our experiments can be described by the flow chart below. We finally designed proprietary probes covering A, B, and DRB1 loci and achieved superior accuracy that exceeded the national standards. Target Gene Purification & Quantification Printing DNA Amplification of PCR Products Microarrays Hybridization s Sample Genotypes & Automated Data Image Scanning & Data Serological Types Reporting Processing UNDERGRADUATE RESEARCH: Feb., 2002 – Jun., 2002: Undergraduate student, Department of Biophysical Science and Technology, Nankai University Bachelor’s thesis: The inhibition of rhein on increase in [Ca2+]i and TNF-release in lipopolysaccharide (LPS)- stimulated macrophages Supervisor: Yang Wenxiu Methods: TNF- production was determined using TNF-sensitive L929 cell line and by MTT method and the kinetics of intracellular free Ca2+ in a single cell were monitored continuously by confocal laser scanning microscope. 3 TSINGHUA UNIVERSITY Research Experience Beijing 100084 P.R.CHINA Rongrong Zhou Results: Rhein had not a distinct effect on TNF-production in normal PM,but could obviously inhibit TNF-release in LPS-stimulated PMin dose-dependent manner, 10-4 mol/L Rhein could inhibit the effect of LPS by 72%. Noticeably, Rhein not only lowered the rising of [Ca2+]i in LPS-stimulated PM, but also altered the dynamical mode of [Ca2+]i, which means that Rhein could transform the ‘plateau-shaped’ peak of [Ca2+]i into the oscillating mode. In the case of extracellular free Ca2+, Rhein further transformed the [Ca2+]i dynamics into a single peak of lower amplitude. These results indicate that Rhein reducing the increase in [Ca2+]i is the key of signal transduction pathway that Rhein inhibited the TNF-production in LPS-stimulated PM It further suggests that inhibition and quasi-periodic modulation on influx of extracelluar calcium as well as on intracellular calcium release is involve in the regulative mechanism of Rhein. National Nature Science Foundation of China Noninvasive Oligonucleotide Microarray for Predicting PIH Qianyong Zhu, Zhi Li, Liang Zhang, Changxu Xu, Bing Chen, Rongrong Zhou and Yong Chen The Third Military Medical University, China & NERCBBT No: 30400480(2004) Abstract: Pregnancy-induced hypertension syndrome (PIH) is a common pregnancy complication, which is a main cause of maternal and newborn morbidity and mortality. Prior to the emergence of clinical symptoms, early prediction and diagnosis and subsequent intervention in time can decrease the incidence and slow the process of this disease greatly. However, all of the predictors currently used in research cannot meet clinical requirements. This project adopted prospective cohort study design. Followed comparison of gene expression pattern in plasma of normal pregnant women and those of PIH patients finally diagnosed in early, midterm and late period of pregnancy was conducted using human total genomic oligonucleotide microarray representing over 23000 pieces of genetic information. Combined bioinformatical methods with comprehensive analysis of clinical data, multiple corresponding genes that can predict the onset of PIH were screened, which were retrospectively confirmed using Real-Time PCR. A fast, accurate, and noninvasive oligonucleotide microarray for predicting PIH was preliminarily devised for screening clinically high-risk or nomal pregnant women. The study is helpful for further investigation in preventing and radical cure of PIH. 4