Analysis of Transgene Expression

Irwin, Michael H.; Pinkert, Carl A.

doi:10.1016/b978-0-12-410490-7.00020-7

Cited by 3 publications

(2 citation statements)

References 29 publications

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“…In vivo fluorescent RNA in situ hybridization (ISH) is a powerful technique utilized to detect the mRNA molecules [1,2] in cells or tissue, enabling researchers to evaluate spatio-temporal gene expression. ISH approaches are popularly utilized in many cytological contexts and are particularly helpful to understand the expression pattern of morphological [3] or stress responsive genes [4] and small RNAs such as lncRNAs or microRNAs [5], that function to regulate various cellular responses.…”

Section: Introductionmentioning

confidence: 99%

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

et al. 2020

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Background: In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatiotemporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-bystep method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of HvGAPDH and the low expressed disease resistance gene Rpg1 in leaf tissue sections of barley and discuss precautions that should be followed during image analysis. Results: We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell associated subsidiary cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. Conclusions: RNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific HvGAPDH and Rpg1 RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis.

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Section: Introductionmentioning

confidence: 99%

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

et al. 2020

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“…In vivo fluorescent RNA in situ hybridization (ISH) is a powerful technique utilized to detect the 46 mRNA molecules [1,2] in cells or tissue, enabling researchers to evaluate spatio-temporal gene 47 expression. ISH approaches are popularly utilized in many cytological contexts and are particularly 48 helpful to understand the expression pattern of morphological [3] or stress responsive genes [4] and small 49 RNAs such as lncRNAs or microRNAs [5], that function to regulate various cellular responses.…”

Section: Introduction 45mentioning

confidence: 99%

Visualization of spatial gene expression in plants by modified RNAscope fluorescentin situhybridization

Solanki

Ameen

Zhao

et al. 2020

Preprint

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The epithelial-dendritic cell interface in pulmonary disease

Ather

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Analysis of Transgene Expression

Cited by 3 publications

References 29 publications

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

Visualization of spatial gene expression in plants by modified RNAscope fluorescentin situhybridization

The epithelial-dendritic cell interface in pulmonary disease

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