RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

Shittu, Mujeeb Olushola; Steenwinkel, Tessa E.; Dion, William; Ostlund, Nathan; Raja, Komal Kumar Bollepogu; Werner, Thomas

doi:10.3390/mps4010020

Cited by 4 publications

(3 citation statements)

References 25 publications

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“…Furthermore, Northern blotting and PCR techniques are unable to determine the localisation of gene expression within cells/tissue. Traditional RNA ISH, which uses digoxigenin (DIG) or radioactive probes, was developed to detect RNA molecules internally based on branched DNA (bDNA) method and principle [ 3 , 6 ]. However, a major limitation of traditional RNA ISH is that it cannot detect other than highly expressed genes, for example H19 (an imprinted maternally expressed transcript), because of the high degree of non-specific binding (lack of specificity) and resultant background noise (poor sensitivity) [ 3 , 7 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the Suitability of RNAscope as a Technique to Measure Gene Expression in Clinical Diagnostics: A Systematic Review

2021

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Objective To evaluate the application of RNAscope in the clinical diagnostic field compared to the current ‘gold standard’ methods employed for testing gene expression levels, including immunohistochemistry (IHC), quantitative real time PCR (qPCR), and quantitative reverse transcriptase PCR (qRT-PCR), and to detect genes, including DNA in situ hybridisation (DNA ISH). Methods This systematic review searched CINAHL, Medline, Embase and Web of Science databases for studies that were conducted after 2012 and that compared RNAscope with one or more of the ‘gold standard’ techniques in human samples. QUADAS-2 test was used for the evaluation of the articles’ risk of bias. The results were reviewed narratively and analysed qualitatively. Results A total of 27 articles (all retrospective studies) were obtained and reviewed. The 27 articles showed a range of low to middle risk of bias scores, as assessed by QUADAS-2 test. 26 articles studied RNAscope within cancer samples. RNAscope was compared to different techniques throughout the included studies (IHC, qPCR, qRT-PCR and DNA ISH). The results confirmed that RNAscope is a highly sensitive and specific method that has a high concordance rate (CR) with qPCR, qRT-PCR, and DNA ISH (81.8–100%). However, the CR with IHC was lower than expected (58.7–95.3%), which is mostly due to the different products that each technique measures (RNA vs. protein). Discussion This is the first systematic review to be conducted on the use of RNAscope in the clinical diagnostic field. RNAscope was found to be a reliable and robust method that could complement gold standard techniques currently used in clinical diagnostics to measure gene expression levels or for gene detection. However, there were not enough data to suggest that RNAscope could stand alone in the clinical diagnostic setting, indicating further prospective studies to validate diagnostic accuracy values, in keeping with relevant regulations, followed by cost evaluation are required. Supplementary Information The online version contains supplementary material available at 10.1007/s40291-021-00570-2.

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Section: Introductionmentioning

confidence: 99%

Evaluation of the Suitability of RNAscope as a Technique to Measure Gene Expression in Clinical Diagnostics: A Systematic Review

2021

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“…ISH was carried out with species-specific RNA probes, as described previously [ 35 ]. Immunohistochemistry for the Y protein in abdomens was performed according to [ 12 ].…”

Section: Methodsmentioning

confidence: 99%

The regulation of a pigmentation gene in the formation of complex color patterns in Drosophila abdomens

et al. 2022

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Changes in the control of developmental gene expression patterns have been implicated in the evolution of animal morphology. However, the genetic mechanisms underlying complex morphological traits remain largely unknown. Here we investigated the molecular mechanisms that induce the pigmentation gene yellow in a complex color pattern on the abdomen of Drosophila guttifera. We show that at least five developmental genes may collectively activate one cis-regulatory module of yellow in distinct spot rows and a dark shade to assemble the complete abdominal pigment pattern of Drosophila guttifera. One of these genes, wingless, may play a conserved role in the early phase of spot pattern development in several species of the quinaria group. Our findings shed light on the evolution of complex animal color patterns through modular changes of gene expression patterns.

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“…We then made DIG-RNA probes by re-amplifying the PCR products from the vector, followed by in vitro transcription. The pupal wings and abdomens were dissected and subjected to hybridization with the probe (1:500) as described previously [27,40]. We stained the pupae using NBT and BCIP solutions from Promega.…”

Section: Rna In Situ Hybridizationmentioning

confidence: 99%

The Genetic Mechanisms Underlying the Concerted Expression of the Yellow and Tan Genes in Complex Patterns on the Abdomen and Wings of Drosophila Guttifera

Raja¹,

Bachman²,

Fernholz³

et al. 2022

Preprint

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How complex morphological patterns form is an intriguing question in developmental biology. However, the mechanisms that generate complex patterns remain largely unknown. Here we sought to identify the genetic mechanisms that regulate the tan (t) gene in a multi-spotted pigmentation pattern on the abdomen and wings of Drosophila guttifera. Previously, we showed that yellow (y) gene expression completely prefigures the abdominal [1] and wing [2] pigment patterns of this species. In the current study, we demonstrate that the t gene is co-expressed with the y gene in nearly identical patterns, both transcripts foreshadowing the adult abdominal and wing melanin spot patterns. We identified cis-regulatory modules (CRMs) of t, one of which drives reporter expression in six longitudinal rows of spots on the developing pupal abdomen, while the second CRM activates the reporter gene in a spotted wing pattern. Comparing the abdominal spot CRMs of y and t, we found a similar composition of putative transcription factor binding sites that are thought to regulate the complex expression patterns of both terminal pigmentation genes y and t. In contrast, the y and t wing spots appear to be regulated by distinct upstream factors. Our results suggest that the D. guttifera abdominal and wing melanin spot patterns have been established through the co-regulation of y and t, shedding light on how complex morphological traits may be regulated through the parallel coordination of downstream target genes.

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RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

Cited by 4 publications

References 25 publications

Evaluation of the Suitability of RNAscope as a Technique to Measure Gene Expression in Clinical Diagnostics: A Systematic Review

Evaluation of the Suitability of RNAscope as a Technique to Measure Gene Expression in Clinical Diagnostics: A Systematic Review

The regulation of a pigmentation gene in the formation of complex color patterns in Drosophila abdomens

The Genetic Mechanisms Underlying the Concerted Expression of the <em>Yellow</em> and <em>Tan</em> Genes in Complex Patterns on the Abdomen and Wings of <em>Drosophila Guttifera</em>

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