Unknown,Transcriptomics,Genomics,Proteomics

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A cost-effective method for double stranded cDNA synthesis for microarray analysis

ABSTRACT: DNA microarrays are two-dimensional arrangements of specific probes deposited on a substrate that have been widely used in gene expression analysis by measuring mRNA accumulation. The use of this type of microarrays involves the synthesis of cDNA, which has to be double stranded (ds) if the microarray probes are of the positive strand. We have used a custom-synthesized non-commercial NimbleGen microarray from melon to evaluate an alternative method of ds cDNA synthesis, which differs substantially in its economical cost relative to a widely recommended method. The results suggested that both methods produce cDNA representative of the melon transcriptome to a similar extent, indicating that the alternative technique provides a cheaper method of ds cDNA synthesis for microarray gene expression assays. Recently, we have analyzed the transcriptome of melon in response to WMV infection. Cotyledons of two genotypes of melon were virus inoculated and transcriptomic responses to the infection were analyzed by comparing infected and mock inoculated samples at 1, 3, and 7 days post-inoculation (dpi). Three biological replicates were performed for each sample. Double stranded cDNA was obtained with the Double stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA), based on the nick translation approach (Mol. Cell. Biol (1982) 2:161-170; Gene (1983) 25:263-269). Raw and processed microarray data are freely available from GEO database under the accession number GSE30111. By using this set of microarray hybridizations as a reference, RNA corresponding to infected cotyledons replicate 3 at 1 dpi (A1) and replicate 1 at 3 dpi (A2) (GEO accession numbers GSM745566 and GSM745567) were used to perform cDNA synthesis by the alternative method (samples B1 and B2, respectively), based on the SMART approach (BioTechniques (2001) 30:892-897), and microarray data were compared.

ORGANISM(S): Cucumis melo

SUBMITTER: Daniel Gonzalez-Ibeas

PROVIDER: E-GEOD-30693 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Virus resistances that are recessively inherited are associated with loss-of-susceptibility resistance alleles. Resistance to Watermelon mosaic virus (WMV) of melon accession TGR-1551 is expressed as a drastic reduction of the virus titer, and is recessively inherited. In this work, viral RNA accumulation was measured in TGR-1551 and in susceptible WMV-infected melon plants by real time quantitative PCR (qPCR), and gene expression of 17,443 unigenes represented in a melon microarray was monitored in a time-course experiment. Virus accumulation was higher in inoculated cotyledons of the resistant genotype up to 7 days post-inoculation; from this time on, virus accumulation was much higher in plants of the susceptible genotype. Microarray experiments were carried with samples from inoculated cotyledons at 1 and 3 dpi to monitor early changes in response to virus infection, and at 7 dpi. Samples from systemically infected leaves harvested at 15 dpi were also included in the analysis. Results showed much more profound transcriptomic alterations in resistant plants compared to susceptible ones. Analyses of gene expression profiles reveal deep and extensive transcriptomic alterations in TGR-1551 plants, many of them involving pathogen response-related genes. Overall, data suggested that resistance to WMV in TGR-1551 is associated with a defense response, contrasting with its recessive nature. Two melon genotypes have been used to analyse transcriptomic responses to infection by Watermelon mosaic virus: Tendral (susceptibel to WMV) and TGR-1551 (resistant to WMV). For each genotype, 60 melon seedlings were inoculated with WMV-M116 and another 60 were mock-inoculated. Cotyledons of 10 plants were harvested at 1, 3, 5, 7, 9 and 15 dpi. At 15 dpi, the systemically infected second true leaf was also harvested. To reduce variability, each biological replicate used in this study was prepared by mixing the RNA extracts from 2 or 4 mock or WMV-inoculated cotyledons, respectively, or from 3 melon leaves. Samples (WMV infected and mock inoculated) corresponding to cotyledons at 1, 3 and 7 dpi, and leaves at 15 dpi were used for microarray hybridisations, three biological replicates for each one, leading to a total of 48 samples.

Project description:Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21â24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analyzed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. This analysis provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melonâvirus interactions. 11 small RNA libraries from several tissues of melon are included en the raw data. 2 samples from ovary, 2 samples from fruit, 1 sample from healthy cotyledons (Cultivar Tendral), 1 samples from healthy cotyledons (genotype TGR-1551), 1 sample from cotyledons (cultivar Tendral) infected with Watermelon mosaic virus (WMV), 1 sample from cotyledons (cultivar TGR-1551) infected with WMV, 1 sample from cotyledons (cultivar Tendral) infected with Melon necrotic spot virus (MNSV, Malfa5 isolate), 1 sample from cotyledons (cultivar Tendral) infected with MNSV (chimeric virus with Malfa5-264 isolates), 1 library from synthetic RNA oligos. Raw reads were obtained from two independent 454 runs, ~22,000 reads each one, to a total of 447,180 reads

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A cost-effective method for double stranded cDNA synthesis for microarray analysis

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