Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies

Lindner, Loïc; Cayrou, Pauline; Jacquot, Sylvie; Birling, Marie‐Christine; Hérault, Yann

doi:10.1016/j.ymeth.2020.07.004

Cited by 24 publications

(23 citation statements)

References 45 publications

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“…RNA isolation and qRT-PCR quantification of tissuederived SIV RNA followed procedures and conditions as described previously [42][43][44][45] and are briefly described here.…”

Section: Rna Extraction and Qpcr Quantification Of Siv Viral Rnamentioning

confidence: 99%

Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian immunodeficiency virus (SIV)

Long¹,

Berkemeier²

2021

Virol J

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Background Simian immunodeficiency virus (SIV)-infected rhesus macaques constitute an excellent model of human HIV infection. Sensitive detection of SIV RNA in cell and tissue samples from infected animals subjected to treatment regimens becomes especially critical in determining which therapeutic attempts are successful, and consequently, which interventions should be prioritized in HIV cure research. Results In this report, we describe the design and testing of a Raindance ddPCR platform-based, sensitive SIV reverse transcription droplet digital PCR (RT-ddPCR) assay by exploring the combinations of various priming conditions and reverse transcriptases, and testing one-step vs. two-step procedures, to eliminate background signal(s) and enable detection and quantification of low level target signals. Conclusions Similar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in RNA samples.

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“…RNA isolation and qRT-PCR quantification of tissuederived SIV RNA followed procedures and conditions as described previously [42][43][44][45] and are briefly described here.…”

Section: Rna Extraction and Qpcr Quantification Of Siv Viral Rnamentioning

confidence: 99%

Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian immunodeficiency virus (SIV)

Long¹,

Berkemeier²

2021

Virol J

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“…2020 [79]. For ddPCR, all primers were designed and synthesized as described in Lindner et al…”

Section: Mouse Rt Droplet Digital Pcr (Ddpcr)mentioning

confidence: 99%

“…2020 [79] excepting Universal Probe Library probe used to Dyrk1a mRNA which is provided by Roche. Dyrk1a and Hprt primers and probes sequences are given in Supplementary Table S6.…”

Section: Mouse Rt Droplet Digital Pcr (Ddpcr)mentioning

confidence: 99%

“…Dyrk1a and Hprt primers and probes sequences are given in Supplementary Table S6. RNA reverse transcription, droplet generation, PCR amplification, droplets quantification and analysis are also described in Lindner et al 2020 [79]. We presented the results as a ratio of the mean of Dyrk1a RNA transcript in Dyrk1a C/C tissue normalized to the mean of Dyrk1a RNA transcript in wt tissue.…”

Section: Mouse Rt Droplet Digital Pcr (Ddpcr)mentioning

confidence: 99%

“…We presented the results as a ratio of the mean of Dyrk1a RNA transcript in Dyrk1a C/C tissue normalized to the mean of Dyrk1a RNA transcript in wt tissue. Experiments were performed following dMIQE guidelines for reporting ddPCR experiments (Supplementary Table S7) [79,80].…”

Section: Mouse Rt Droplet Digital Pcr (Ddpcr)mentioning

confidence: 99%

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Dyrk1agene dosage in glutamatergic neurons has key effects in cognitive deficits observed in mouse models of MRD7 and Down syndrome

Brault

Nguyen

Flores-Gutiérrez

et al. 2021

Preprint

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Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Mutation in the DYRK1A gene located on human chromosome 21 (Hsa21) leads to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles (MRD7). Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS). Dyrk1a is expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact of Dyrk1a gene copy number variation in glutamatergic neurons using a conditional knockout allele of Dyrk1a crossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence of Dyrk1a dosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall, Dyrk1a dosage in glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed that Dyrk1a is involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach.Author summaryThe Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A, DYRK1A, drives cognitive alterations with increased dose in Down syndrome (DS) or with reduced dose in mental retardation disease 7 (MRD7). Here we report that specific and complete loss of Dyrk1a in glutamatergic neurons induced a range of specific cognitive phenotypes and alter the expression of genes involved in neurotransmission in the hippocampus. We further explored the consequences of Dyrk1a dosage in glutamatergic neurons on the cognitive phenotypes observed respectively in MRD7 and DS mouse models and we found specific roles in long-term explicit memory with no impact on motor activity, short-term working memory, and susceptibility to epilepsy. Then we demonstrated that DYRK1A is a component of the glutamatergic post-synapse and interacts with several component such as NR2B and PSD95. Altogether our work describes a new role of DYRK1A at the glutamatergic synapse that must be considered to understand the consequence of treatment targeting DYRK1A in disease.

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CRISMERE Chromosome Engineering in Mouse and Rat

Schaeffer

Lindner

Hérault

et al. 2023

Methods in Molecular Biology

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Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies

Cited by 24 publications

References 45 publications

Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian immunodeficiency virus (SIV)

Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian immunodeficiency virus (SIV)

Dyrk1agene dosage in glutamatergic neurons has key effects in cognitive deficits observed in mouse models of MRD7 and Down syndrome

CRISMERE Chromosome Engineering in Mouse and Rat

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