Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`- Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)

This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RN

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

Abstract Background Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. Conclusion The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.This work was supported by Research Grant Award No. IS-4223-09C from BARD, the United States-Israel Binational Agricultural Research and Development Fund, and by SNC Laboratoire ASL, de Ruiter Seeds B.V., Enza Zaden B.V., Gautier Semences S.A., Nunhems B.V., Rijk Zwaan B.V., Sakata Seed Inc, Semillas Fitó S.A., Seminis Vegetable Seeds Inc, Syngenta Seeds B.V., Takii and Company Ltd, Vilmorin and Cie S.A. and Zeraim Gedera Ltd (all of them as part of the support to ICuGI). CC was supported by CNRS ERL 8196.Peer Reviewe

A Cost-effective Double-Stranded cDNA Synthesis for Plant Microarrays

[EN] 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 melon custom-synthesized noncommercial NimbleGen microarray to evaluate a modification of the SMART¿ (switching mechanism at the 5¿ end of the RNA transcript) procedure of ds cDNA synthesis, which differs substantially in its economical cost relative to a widely recommended method based on the nick translation approach. The results suggested that both methods produce cDNA representative of the transcriptome to a similar extent, indicating that the alternative technique provides a cheaper method of ds cDNA synthesis for plant microarray gene expression assays when the RNA starting material is not limiting. © 2012 Springer-Verlag.This work was supported by grant AGL2009-07552/AGR. We thank Mari Carmen Montesinos and Blanca Gosalvez for their technical assistance.Gonzalez-Ibeas, D.; Blanca Postigo, JM.; Cañizares Sales, J.; Truniger, V.; Aranda, MA. (2012). A Cost-effective Double-Stranded cDNA Synthesis for Plant Microarrays. Plant Molecular Biology Reporter. 30(5):1276-1282. https://doi.org/10.1007/s11105-012-0427-5S12761282305Barbulovic-Nad I, Lucente M, Sun Y, Zhang M, Wheeler AR, Bussmann M (2006) Bio-microarray fabrication techniques—a review. Crit Rev Biotechnol 26:237–259Barrett T, Troup DB, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Muertter RN, Holko M, Ayanbule O, Yefanov A, Soboleva A (2011) NCBI GEO: archive for functional genomics data sets—10 years on. Nucleic Acids Res 39:D1005–D1010Carvalho B, Bengtsson H, Speed TP, Irizarry RA (2007) Exploration, normalization, and genotype calls of high-density oligonucleotide SNP array data. Biostatistics 8:485–499Dafforn A, Chen P, Deng G, Herrler M, Iglehart D, Koritala S, Lato S, Pillarisetty S, Purohit R, Wang M, Wang S, Kurn N (2004) Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis. Biotechniques 37:854–857Dudoit S, Gentleman RC, Quackenbush J (2003) Open source software for the analysis of microarray data. Biotechniques, Suppl:45–51Gonzalez-Ibeas D, Blanca J, Roig C, Gonzalez-To M, Pico B, Truniger V, Gomez P, Deleu W, Cano-Delgado A, Arus P, Nuez F, Garcia-Mas J, Puigdomenech P, Aranda M (2007) MELOGEN: an EST database for melon functional genomics. BMC Genomics 8:306Gonzalez-Ibeas D, Cañizares J, Aranda MA (2011) Microarray analysis shows that recessive resistance to Watermelon mosaic virus in melon is associated with the induction of defense response genes. Mol Plant-Microbe Interact 25:107–118Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263–269Lueders T, Friedrich MW (2003) Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl Environ Microbiol 69:320–326Mascarell-Creus A, Cañizares J, Vilarrasa-Blasi J, Mora-García S, Blanca J, Gonzalez-Ibeas D, Saladié M, Roig C, Deleu W, Picó-Silvent B, López-Bigas N, Aranda MA, Garcia-Mas J, Nuez F, Puigdomènech P, Caño-Delgado AI (2009) An oligo-based microarray offers novel transcriptomic approaches for the analysis of pathogen resistance and fruit quality traits in melon (Cucumis melo L.). BMC Genomics 10:467Mathieu-Daudé F, Welsh J, Vogt T, McClelland M (1996) DNA rehybridization during PCR: the “Cot effect” and its consequences. Nucleic Acids Res 24:2080–2086Matz M, Shagin D, Bogdanova E, Britanova O, Lukyanov S, Diatchenko L, Chenchik A (1999) Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res 27:1558–1560Nygaard V, Løland A, Holden M, Langaas M, Rue H, Liu F, Myklebost O, Fodstad Ø, Hovig E, Smith-Sørensen B (2003) Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance. BMC Genomics 4:11Okayama H, Berg P (1982) High-efficiency cloning of full-length cDNA. Mol Cell Biol 2:161–170Page GP, Coulibaly I (2008) Bioinformatic tools for inferring functional information from plant microarray data: tools for the first steps. Int J Plant Genomics 2008:147563Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol 64:3724–3730Puskás LG, Zvara A, Hackler L Jr, Van Hummelen P (2002) RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 32:1330–1340R Development Core Team. R (2010) A language and environment for statistical computing. Vienna, AustriaRaychaudhuri S, Stuart JM, Altman RB (2000) Principal components analysis to summarize microarray experiments: application to sporulation time series. Pac Symp Biocomput 2000:455–466Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, Li J, Thiagarajan M, White JA, Quackenbush J (2006) TM4 microarray software suite. Methods Enzymol 411:134–193Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467–470Shagin DA, Rebrikov DV, Kozhemyako VB, Altshuler IM, Shcheglov AS, Zhulidov PA, Bogdanova EA, Staroverov DB, Rasskazov VA, Lukyanov S (2002) A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res 12:1935–1942Soria-Guerra RE, Rosales-Mendoza S, Gasic K, Wisniewski ME, Band M, Korban SS (2011) Gene expression is highly regulated in early developing fruit of apple. Plant Mol Biol Rep 29:885–897Stolf-Moreira R, Lemos EGM, Carareto-Alves L, Marcondes J, Pereira SS, Rolla AAP, Pereira RM, Neumaier N, Binneck E, Abdelnoor RV, Oliveira MCN, Marcelino FC, Farias JRB, Nepomuceno AL (2010) Transcriptional profiles of roots of different soybean genotypes subjected to drought stress. Plant Mol Biol Rep 29:19–34Suzuki MT, Giovannoni SJ (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol 62:625–630Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD, Eberwine JH (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A 87:1663–1667Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM (2000) High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 18:457–459Yang G, Zhou R, Tang T, Chen X, Ouyang J, He L, Li W, Chen S, Guo M, Li X, Shi S (2010) Gene expression profiles in response to salt stress in Hibiscus tiliaceus. Plant Mol Biol Rep 29:609–617Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30:892–89

Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`- Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)

This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RNAThis article is from Bio-protocol 5 (2015): e1386. Posted with permission.</p

Analysis of the interaction melon-melon necrotic spot virus controlled by the recessive resistance gene Nsv

International audienc

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

Background:Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. - Result: We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full length enriched cDNA clones that were sequenced from both ends. Analysis of these full length transcripts indicated that sizes of melon 5'and 3'UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. Conclusion: The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker assisted breeding of melon and closely relate species, comparative genomic studies and for gaining insights into gene expression patterns