Unknown,Transcriptomics,Genomics,Proteomics

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An Easy Operating Pathogen Microarray (EOPM) Platform for Rapid Screening of Vertebrate Pathogens

ABSTRACT: An Easy Operating Pathogen Microarray (EOPM) was designed to detect almost all known pathogens and related species based on their genomic sequences. For effective identification of pathogens from EOPM data, a statistical enrichment algorithm has been proposed and further implemented in a user-friendly interface. A microarray was designed with probes for vertebrate-infecting virus sequences in EMBL, 18S rRNA fungi and parasite sequences from EMBL, and 16S rRNA sequences of bacteria from RDP, synthesized on the Agilent platform. The array was tested using 2 color dyes on cultured microbes and on clinical samples from sick and healthy people, looking for differences in clinically ill people compared to a number of healthy "controls".

ORGANISM(S): Homo sapiens

SUBMITTER: wang liang

PROVIDER: E-GEOD-45860 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Singapore grouper iridovirus (SGIV) is the major agent that causes severe iridovirus diseases in grouper maricluture. Based on the genomic information, a DNA microarray, containing probes corresponding to 162 putative SGIV open reading frames (ORFs), was constructed to map the viral gene transcriptional profiles over the time course by establishing the models of SGIV-infected GS cells and SGIV-infected grouper. All the data from real-time RT-PCR, RT-PCR and dilution RT-PCR assays were confirmed with the findings of microarrays, which were clustered into groups with the similarity expression profiles by the Self-Organizing Maps (SOMs) approach. The microarray analysis showed that SGIV had big differential expression profiles in the special infected cells and organ and the viral DNA replication mechanisms were firstly prevented as an important strategy of the host defense during the natural course infection.Our studies firstly uncover the relative of a marine viral gene expression patterns between in vitro and in vivo infection, which provides a better understanding of SGIV transcription regulation and a greater degree shared with other iridoviruses on their repliaction and pathogenesis. Keywords: time course To further characterize SGIV gene expression patterns and to monitor the gene temporal kinetic transcription program on a genome-wide scale, we monitored viral gene expression profiles in vitro and in vivo infection.For the experiment of infection in vitro, GS cell monolayers cultured in 75cm2 flask were inoculated with 1ml of SGIV (5.0 Ã105.5 TCID50/ml) at 25oC. The mock-infected cells (as reference samples) were treated in the same manner as SGIV-infected cells but with fresh culture medium. Total RNA was isolated from the cells at 1, 2, 4, 6, 8, 10, 12, 16, 24, 36, 48, 72, and 96 hours post-infection (h p.i.), respectively. For the experiment of infection in vivo, Grouper Epinephelus tauvina juveniles with approximately 40-50 g were experimentally infected with 150 Âµl of the SGIV inoculum (5.0Ã105.5 TCID50/ml) and held in tanks supplied with running seawater at 25oC. Control fishes were injected with the same volume of EMEM. Total RNA was harvested from the spleens of 5 fishes randomly selected from the experimental population at 1, 2, 3, 4, 5, 7, 9, 11, and 15 days post-infection (d p.i.). Total RNA from the spleens of 35 mock-infected fishes was used as reference samples. After visual inspection for the presence of image artifacts, such as scratches, dirt, contamination, high region, or overall background on the array, the scanned images were saved as TIF files and further analyzed to generate raw data using SpotDataTM software (CapitalBio). After filtering the low-intensity spots and background-noise value, a global scaling procedure was performed to normalize among the different arrays and the different channels of same arrays using housekeeping gene of piscine 18S RNA which was also spotted in triplicate. To estimate the variance result from dye-bias, a swap-dye strategy was used for the virus-infected and mock-infected samples at 48 h p.i.

Project description:Radix bupleuri is a traditional Chinese crude drug and it is still widely used in contemporary medicine. Bupleurum chinense DC. and B. scorzonerifolium Willd. are officially recorded as the source species of Radix bupleuri in Pharmacopoeia of the PeopleM-bM-^@M-^Ys Republic of China. The obvious difference in chemical active components and pharmacological action of Radix bupleuri derived from the two species has caused attention. But to date, no definite criterions and regulates are issued based on plant species sources to direct the standard uses of Radix bupleuri for their extracts and traditional decoction pieces in medical market. Using 454-FLX pyrosequencing with one half-plate run for each cDNA library, a total of 419,576 and 416,112 reads, among those 313,483 and 342,263 high quality ESTs were obtained for B. chinense and B. scorzonerifolium, respectively. The average read length was 279 bp and 310 bp, respectively. The assembled 28,897 and 30,912 unigenes for B. chinense and B. scorzonerifolium were successively annotated based on sequence similarity by major public nucleotide/protein databases, Nt, Nr, SwissProt, KEGG, COG, Interpro and GO. Based on the alignments, 71.9% (20,787) and 75.8% (23,426) of the total unigenes for B. chinense and B. scorzonerifolium could be matched to nucleotides from NCBI Nt database. Certain percent from 27.1% (7,820) to 70.8% (20,461) of the total unigenes for B. chinense, and from 26.4% (8,145) to 72.1% (22,289) of the total unigenes for B. scorzonerifolium, could be matched to proteins from different databases. For the same database, similar percent of the total unigenes for B. chinense and B. scorzonerifolium were annotated. A total of 17,117 (59.2%) and 19,416 (62.8%) unigenes for B. chinense and B. scorzonerifolium could be found homologous genes in opposite dataset. According to KEGG annotation, 3,637 and 3,821 unigenes were assigned relating to metabolism in datasets of B. chinense and B. scorzonerifolium, respectively. Carbohydrate metabolism, energy metabolism and amino acid metabolism were the highest three groups in metabolism category and followed by lipid metabolism group for B. chinense. In comparison, lipid metabolism group had most unigenes for B. scorzonerifolium and it had much more unigenes than the same group for B. chinense (827 to 562). A total of 70 and 52 unigenes were related to metabolism of terpenoids and polyketides in two separate datasets, which seems agree with the situation of higher content of terpenoid saikosaponins in B. chinense than B. scorzonerifolium in some extent. To compare the biosynthesis of main bioactive components in roots of B. chinense and B. scorzonerifolium, putative genes which may participate in the biosynthesis of terpenoid, triterpenoid, sterol, ligan and flavonoid were searched according to unigenes annotation. Most lignan and flavonoid biosynthesis related genes were found in both datasets of B. chinense and B. scorzonerifolium, while some terpenoid or triterpenoid related genes were dominantly found in the dataset of B. chinense. This reflected in some extent that these genes expressed in low levels and relative metabolites were biosynthesized in lower quantities in roots of B. chinense than those of B. scorzonerifolium. Besides abovementioned comparison on two transcriptome, the expression of some genes involved in biosyntheses of main medicinal secondary metabolites were analyzed by qRT-PCR in roots of the two species. These molecular biological data will make a useful clue and arouse more studies on the reasonable application of the two Bupleurum species. The fine roots of two-year old plants of B. chinense and B. scorzonerifolium planted in the same place at the same time were collected at the usual harvest time of medicinal materials of Bupleurum. Those roots from the two species plants were used for 454 sequencing.

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An Easy Operating Pathogen Microarray (EOPM) Platform for Rapid Screening of Vertebrate Pathogens

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