Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A gene expression microarray for Nicotiana benthamiana based on de novo transcriptome sequence assembly

ABSTRACT: Gene expression microarray was designed, based on two recently published versions of the N. benthamiana transcriptome (v.3 and v.5). Based on homology searches and hybridization tests we identified the sense strand in 106,684 transcriptome contigs and used this information to design a Nb-105k microarray on Agilent eArray platform. By the hybridization of N. benthamiana roots vs leaves RNA samples we showed high specificity and sensitivity of the new microarray in detection of differentially expressed transcripts. Agilent microarrays in 2x105k format (two microarrays per slide) were used for hybridization of labeled RNA from leaves vs roots of N. benthamiana plants. Four biological replicates were used for two-color design in a dye-swap manner.

ORGANISM(S): Nicotiana benthamiana

SUBMITTER: Agnieszka Zmienko

PROVIDER: E-GEOD-76631 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation-based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs were the same in different plant species and in the absence of RDR6. We used the TerminatorTM 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were not complementary to highly abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double-stranded RNA and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double-stranded RNA or by RNA-dependent RNA polymerase. Size-fractionated (19-24 nt) small RNA from early systemic leaves of CymRSV-infected Nicotiana benthamiana total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification, the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.

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A gene expression microarray for Nicotiana benthamiana based on de novo transcriptome sequence assembly

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