A new PCR-based approach for the preparation of RNA probe

Suzuki, Takuya; Akimoto, Masayuki; Mandai, Michiko; Takahashi, Masayo; Yoshimura, Nagahisa

doi:10.1016/j.jbbm.2004.12.003

Cited by 8 publications

(6 citation statements)

References 18 publications

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“…Sense and antisense riboprobes were synthesized using a PCR-based in situ hybridization technique as previously described (Suzuki et al, 2005;Young et al, 1993). Briefly, PCR was performed with TTLL3 gene-specific primers encompassing a T7 RNA polymerase-binding site.…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Grau

Masson

Gadadhar

et al. 2017

Journal of Cell Science

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Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are nonmotile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina. Ttll3−/− mice lack glycylation in photoreceptors, which results in shortening of connecting cilia and slow retinal degeneration. Moreover, absence of glycylation results in increased levels of tubulin glutamylation in photoreceptors, and inversely, the hyperglutamylation observed in the Purkinje cell degeneration ( pcd) mouse abolishes glycylation. This suggests that both posttranslational modifications compete for modification sites, and that unbalancing the glutamylation-glycylation equilibrium on axonemes of connecting cilia, regardless of the enzymatic mechanism, invariably leads to retinal degeneration.

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Section: In Situ Hybridizationmentioning

confidence: 99%

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Grau

Masson

Gadadhar

et al. 2017

Journal of Cell Science

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“…To visualize the bound signals, samples were incubated with BM‐purple (Roche Applied Science, 11 442 074 001) for 2‐3 hr. The RNA probes were generated using a PCR based method described previously . Briefly, T7 promoter sequences were introduced to the 5‐prime end of the reverse and forward primers, enabling the synthesis of antisense and sense transcripts, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The RNA probes were generated using a PCR based method described previously. 40 Briefly, T7 promoter sequences were introduced to the 5-prime end of the reverse and forward primers, enabling the synthesis of antisense and sense transcripts, respectively. PCR fragments were then amplified using gene-specific primers (Table 1), followed by generation of the digoxigenin labeled riboprobes using T7 RNA polymerase (Beyotime, D7069) and DIG RNA labeling mix (Roche Applied Science, 11 277 073 910).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Dennd1a, a susceptibility gene for polycystic ovary syndrome, is essential for mouse embryogenesis

Shi

Gao

Cao

et al. 2019

Developmental Dynamics

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Background The DENND1A has been identified as a guanine nucleotide exchange factor for small GTPase Rab35, which functions in endocytic trafficking to mediate the recycling of selective cargos. Genetic alterations within the DENND1A gene have been implicated in human disease such as polycystic ovary syndrome (PCOS). However, the role of DENND1A in developmental and reproductive processes is largely unknown. Results Using Dennd1a gene knockout mice, we uncovered that homogeneous Dennd1a−/− mutants died around embryonic day (E) 14.5. The brain of Dennd1a−/− embryos exhibited defects, partially attributed to the dysregulation of cell division and survival in the telencephalon. The transcription of Fgf8 mRNA was ectopically elevated in the dorsal midline of telencephalon, concomitant with a decrease of active β‐catenin and Axin2 in the brain of Dennd1a−/− embryos. During liver morphogenesis, the ablation of Dennd1a impaired hepatic cell proliferation, the differentiation of hepatocyte, and hepatic hematopoiesis. In addition, loss of Dennd1a also affected the development of primordial germ cells. Conclusions We demonstrate that Dennd1a, a susceptibility gene for PCOS, is essential for embryogenesis, probably through the mediation of endocytic recycling of selective cargos that are involved in cell signaling crucial for the development of multiple embryonic organ systems.

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“…Following the initial screening, the selected primer pairs were then tested against the representative paramyxoviruses listed in Table 1. Finally, to test for unanticipated nonspecific reactivity, the PCR assays were tested against pooled nucleic acids of influenza A and B viruses, rhinoviruses, adenovirus, two distinct human coronaviruses, human coronavirus The sensitivities of the PCR assays were determined using two sources of RNA: RNA that was extracted from each dilution of a 10-fold dilution series of virus-infected cell culture with known infectivity titers (PFU) and serial dilutions of synthetic RNA that was transcribed in vitro from cloned genome fragments as previously described (18).…”

Section: Rt-pcr and Nested Rt-pcr Amplificationmentioning

confidence: 99%

Sensitive and Broadly Reactive Reverse Transcription-PCR Assays To Detect Novel Paramyxoviruses

et al. 2008

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We have developed a set of reverse transcription-PCR assays for the detection and identification of known and novel paramyxoviruses in clinical specimens. Primers were designed from the conserved motifs of the polymerase pol gene sequences to detect members of the Paramyxovirinae or Pneumovirinae subfamily or groups of genera within the Paramyxovirinae subfamily. The consensus-degenerate hybrid oligonucleotide primer design and seminested or nested PCR assay design were used to enhance the breadth of reactivity and sensitivity of the respective assays. Using expressed RNA and 10-fold dilution series of virus-infected tissue culture isolates from different members of the family or genera, these assays were able to detect on average between 100 and 500 copies of template RNA. The assays were specific to the respective group of genera or subfamily viruses. This set of primers enhances our ability to look for novel viruses in outbreaks and diseases of unknown etiology.

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A new PCR-based approach for the preparation of RNA probe

Cited by 8 publications

References 18 publications

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Dennd1a, a susceptibility gene for polycystic ovary syndrome, is essential for mouse embryogenesis

Sensitive and Broadly Reactive Reverse Transcription-PCR Assays To Detect Novel Paramyxoviruses

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