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Complementarity of medium-throughput in situ RNA hybridization and tissue-specific transcriptomics: case study of Arabidopsis seed development kinetics.

Francoz E, Ranocha P, Pernot C, Le Ru A, Pacquit V, Dunand C, Burlat V - Sci Rep (2016)

Bottom Line: Integration of results illustrates the complementarity of both datasets.Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other.For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326 Castanet Tolosan, France.

ABSTRACT
The rationale of this study is to compare and integrate two heterologous datasets intended to unravel the spatiotemporal specificities of gene expression in a rapidly growing and complex organ. We implemented medium-throughput RNA in situ hybridization (ISH) for 39 genes mainly corresponding to cell wall proteins for which we have particular interest, selected (i) on their sequence identity (24 class III peroxidase multigenic family members and 15 additional genes used as positive controls) and (ii) on their expression levels in a publicly available Arabidopsis thaliana seed tissue-specific transcriptomics study. The specificity of the hybridization signals was carefully studied, and ISH results obtained for the 39 selected genes were systematically compared with tissue-specific transcriptomics for 5 seed developmental stages. Integration of results illustrates the complementarity of both datasets. The tissue-specific transcriptomics provides high-throughput possibilities whereas ISH provides high spatial resolution. Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other. For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

No MeSH data available.


Related in: MedlinePlus

Thresholds of tissue-specific transcriptomic values compatible with ISH are specific to each developmental stage/tissue.For each developmental stage/tissue tissue-specific transcriptomic sample ((a,b) globular developmental stage; (c,d) mature green developmental stage; (a,c) embryo proper tissue; (b,d) seed coat tissue), the 23,933 genes present on the microarray were classified according to their tissue-specific transcriptomic expression values. The range 0–44 corresponded to the expression values below the 45 detection limit of the transcriptomic study (55–65% of the genes); the range 45–299 corresponded to the expression values between the 45 detection limit of the transcriptomics and the 300 arbitrary cut-off that we initially defined for our ISH study; the other ranges were arbitrarily set to allow the distribution of the genes in various expression value groups. The resulting number of genes within each range was plotted on individual histograms. All the genes analysed by ISH in this study were positioned above the histograms according to their individual tissue-specific transcriptomic expression value. The ISH results were colour-coded in red/orange/white as in Fig. 2, according to Supplementary Figs. S4–S42. The deducted total number of genes compatible, to various extend, with ISH was posted on the top of each graph within double arrows using shades of the same colour coding. This clearly illustrates that the sensitivity of ISH is dependent on spatiotemporal parameters and the complementarity of transcriptomics and ISH. The corresponding histograms for all developmental stages and tissues are available in Supplementary Figs. S46–S51.
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f3: Thresholds of tissue-specific transcriptomic values compatible with ISH are specific to each developmental stage/tissue.For each developmental stage/tissue tissue-specific transcriptomic sample ((a,b) globular developmental stage; (c,d) mature green developmental stage; (a,c) embryo proper tissue; (b,d) seed coat tissue), the 23,933 genes present on the microarray were classified according to their tissue-specific transcriptomic expression values. The range 0–44 corresponded to the expression values below the 45 detection limit of the transcriptomic study (55–65% of the genes); the range 45–299 corresponded to the expression values between the 45 detection limit of the transcriptomics and the 300 arbitrary cut-off that we initially defined for our ISH study; the other ranges were arbitrarily set to allow the distribution of the genes in various expression value groups. The resulting number of genes within each range was plotted on individual histograms. All the genes analysed by ISH in this study were positioned above the histograms according to their individual tissue-specific transcriptomic expression value. The ISH results were colour-coded in red/orange/white as in Fig. 2, according to Supplementary Figs. S4–S42. The deducted total number of genes compatible, to various extend, with ISH was posted on the top of each graph within double arrows using shades of the same colour coding. This clearly illustrates that the sensitivity of ISH is dependent on spatiotemporal parameters and the complementarity of transcriptomics and ISH. The corresponding histograms for all developmental stages and tissues are available in Supplementary Figs. S46–S51.

Mentions: The Supplementary Table S1 was sequentially used to distribute, for each of 36 tissue-specific transcriptomic samples (all samples except the 6 whole seed samples), the 23,933 genes within 10 groups according to their transcriptomic expression value. The groups corresponded to expression values of 0–44 (below the detection limit of the transcriptomic study; 55–65% of the genes depending on the sample), 45–299 (below the arbitrary 300 cut-off used for our first selection of candidate genes), 1000–1999, 2000–2999, 3000–3999, 4000–4999, 5000–9999, 10000–19999, 20000-max. In order to fully integrate our ISH results with the whole tissue-specific transcriptomic data, the abbreviation of the 37 genes of the 39 genes studied by ISH that were present on the array (all 39 genes except AtPRX13 and AtPRX32) was positioned on the individual histograms on the top of the range corresponding to their tissue-specific transcriptomic value (Fig. 3; Supplementary Figs. S46–S51). In order to summarize the ISH results, we kept the same red/orange/white colour code described above. Less genes were studied in ISH for the preglobular stage which was under-represented on the tissue arrays. We finally calculated within colour-coded double arrows positioned on the top of the histograms, the sum of genes for which ISH could be sensitive enough to various degrees for each tissue/developmental stage.


Complementarity of medium-throughput in situ RNA hybridization and tissue-specific transcriptomics: case study of Arabidopsis seed development kinetics.

Francoz E, Ranocha P, Pernot C, Le Ru A, Pacquit V, Dunand C, Burlat V - Sci Rep (2016)

Thresholds of tissue-specific transcriptomic values compatible with ISH are specific to each developmental stage/tissue.For each developmental stage/tissue tissue-specific transcriptomic sample ((a,b) globular developmental stage; (c,d) mature green developmental stage; (a,c) embryo proper tissue; (b,d) seed coat tissue), the 23,933 genes present on the microarray were classified according to their tissue-specific transcriptomic expression values. The range 0–44 corresponded to the expression values below the 45 detection limit of the transcriptomic study (55–65% of the genes); the range 45–299 corresponded to the expression values between the 45 detection limit of the transcriptomics and the 300 arbitrary cut-off that we initially defined for our ISH study; the other ranges were arbitrarily set to allow the distribution of the genes in various expression value groups. The resulting number of genes within each range was plotted on individual histograms. All the genes analysed by ISH in this study were positioned above the histograms according to their individual tissue-specific transcriptomic expression value. The ISH results were colour-coded in red/orange/white as in Fig. 2, according to Supplementary Figs. S4–S42. The deducted total number of genes compatible, to various extend, with ISH was posted on the top of each graph within double arrows using shades of the same colour coding. This clearly illustrates that the sensitivity of ISH is dependent on spatiotemporal parameters and the complementarity of transcriptomics and ISH. The corresponding histograms for all developmental stages and tissues are available in Supplementary Figs. S46–S51.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837347&req=5

f3: Thresholds of tissue-specific transcriptomic values compatible with ISH are specific to each developmental stage/tissue.For each developmental stage/tissue tissue-specific transcriptomic sample ((a,b) globular developmental stage; (c,d) mature green developmental stage; (a,c) embryo proper tissue; (b,d) seed coat tissue), the 23,933 genes present on the microarray were classified according to their tissue-specific transcriptomic expression values. The range 0–44 corresponded to the expression values below the 45 detection limit of the transcriptomic study (55–65% of the genes); the range 45–299 corresponded to the expression values between the 45 detection limit of the transcriptomics and the 300 arbitrary cut-off that we initially defined for our ISH study; the other ranges were arbitrarily set to allow the distribution of the genes in various expression value groups. The resulting number of genes within each range was plotted on individual histograms. All the genes analysed by ISH in this study were positioned above the histograms according to their individual tissue-specific transcriptomic expression value. The ISH results were colour-coded in red/orange/white as in Fig. 2, according to Supplementary Figs. S4–S42. The deducted total number of genes compatible, to various extend, with ISH was posted on the top of each graph within double arrows using shades of the same colour coding. This clearly illustrates that the sensitivity of ISH is dependent on spatiotemporal parameters and the complementarity of transcriptomics and ISH. The corresponding histograms for all developmental stages and tissues are available in Supplementary Figs. S46–S51.
Mentions: The Supplementary Table S1 was sequentially used to distribute, for each of 36 tissue-specific transcriptomic samples (all samples except the 6 whole seed samples), the 23,933 genes within 10 groups according to their transcriptomic expression value. The groups corresponded to expression values of 0–44 (below the detection limit of the transcriptomic study; 55–65% of the genes depending on the sample), 45–299 (below the arbitrary 300 cut-off used for our first selection of candidate genes), 1000–1999, 2000–2999, 3000–3999, 4000–4999, 5000–9999, 10000–19999, 20000-max. In order to fully integrate our ISH results with the whole tissue-specific transcriptomic data, the abbreviation of the 37 genes of the 39 genes studied by ISH that were present on the array (all 39 genes except AtPRX13 and AtPRX32) was positioned on the individual histograms on the top of the range corresponding to their tissue-specific transcriptomic value (Fig. 3; Supplementary Figs. S46–S51). In order to summarize the ISH results, we kept the same red/orange/white colour code described above. Less genes were studied in ISH for the preglobular stage which was under-represented on the tissue arrays. We finally calculated within colour-coded double arrows positioned on the top of the histograms, the sum of genes for which ISH could be sensitive enough to various degrees for each tissue/developmental stage.

Bottom Line: Integration of results illustrates the complementarity of both datasets.Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other.For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326 Castanet Tolosan, France.

ABSTRACT
The rationale of this study is to compare and integrate two heterologous datasets intended to unravel the spatiotemporal specificities of gene expression in a rapidly growing and complex organ. We implemented medium-throughput RNA in situ hybridization (ISH) for 39 genes mainly corresponding to cell wall proteins for which we have particular interest, selected (i) on their sequence identity (24 class III peroxidase multigenic family members and 15 additional genes used as positive controls) and (ii) on their expression levels in a publicly available Arabidopsis thaliana seed tissue-specific transcriptomics study. The specificity of the hybridization signals was carefully studied, and ISH results obtained for the 39 selected genes were systematically compared with tissue-specific transcriptomics for 5 seed developmental stages. Integration of results illustrates the complementarity of both datasets. The tissue-specific transcriptomics provides high-throughput possibilities whereas ISH provides high spatial resolution. Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other. For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

No MeSH data available.


Related in: MedlinePlus