Regulation of prefrontal cortex myelination by the microbiota methods
Aim. Evidence-backed execution summary for Regulation of prefrontal cortex myelination by the microbiota methods from Regulation of prefrontal cortex myelination by the microbiota.
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This experiment, in seven questions
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Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Quantitative real-time PCR
reagent used in the protocol.
- Use
- RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene expression was analysed using TaqMan Gene Expression Assays ( ) on an AB7300 system (Applied Biosystems, Thermo Fisher Scientific). Express...
Protein extraction and western blot
reagent used in the protocol.
- Use
- Total protein was extracted using a commercially available mirVana PARIS RNA and Native Protein Purification kit (Thermo Fisher Scientific). Protein levels were detected using appropriate primary antibody dilutions against MOG (1:1000; sourced from Abcam, ab109746) and secondary antibody (Alexa 594-conjugated antibo...
Transmission electron microscopy
reagent used in the protocol.
- Use
- Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK)...
Tissue extraction and RNA sequencing
Several key brain regions (amygdala, PFC, striatum, cerebellum, hippocampus and frontal cortex) were rapidly hand-dissected and stored at 4 °C in RNAlater (Sigma-Aldrich, Wicklow, Ireland) for 24 h followed by storage at -80 °C until tissue processing. RNA sequencing was performed a...
- Use
- Several key brain regions (amygdala, PFC, striatum, cerebellum, hippocampus and frontal cortex) were rapidly hand-dissected and stored at 4 °C in RNAlater (Sigma-Aldrich, Wicklow, Ireland) for 24 h followed by storage at -80 °C until tissue processing. RNA sequencing was performed a...
Quantitative real-time PCR
RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene expression was analysed using TaqMan Gene Expression Assays ( ) on an AB7300 system (Applied Biosystems, Thermo Fisher Scientific). Express...
- Use
- RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene expression was analysed using TaqMan Gene Expression Assays ( ) on an AB7300 system (Applied Biosystems, Thermo Fisher Scientific). Express...
Transmission electron microscopy
Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK)...
- Use
- Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK)...
Statistics
All statistics used for analysis of differential gene expression were according to the description of R package DESeq2 with default parameters. Multiple-testing corrections were performed for all analyses, the adjusted P -value ( P adj ) was calculated using the Benjamini-Hochberg procedure (also known as fals...
- Use
- All statistics used for analysis of differential gene expression were according to the description of R package DESeq2 with default parameters. Multiple-testing corrections were performed for all analyses, the adjusted P -value ( P adj ) was calculated using the Benjamini-Hochberg procedure (also known as fals...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All statistics used for analysis of differential gene expression were according to the description of R package DESeq2 with default parameters. Multiple-testing corrections were performed for all analyses, the adjusted P -value ( P adj ) was calculated using the Benjamini-Hochberg procedure (also known as fals...
Before you run
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First confirmation
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Open the source paper before finalizing run-specific details.
Procurement checkpoint
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Open quote workflowStep-by-step procedure
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Materials and methods
GF and CON Swiss Webster breeding pairs were obtained from Taconic (Germantown, NY, USA). GF mice were housed in gnotobiotic isolators under a strict 12-h light/dark cycle in cages of 4-5 animals. On postnatal day 21, just after weaning, exGF mice were taken from isolators and housed next to CON mice. CON and exGF mice were housed in the standard animal unit, which allows for exGF mice to be exposed to environmental microbes resulting in colonization. CON mice are similarly housed under regulated conditions (temperature 20-21 °C, 55-60% humidity), under the same 12-h light/dark cycle and housed 4-5 per cage. Autoclaved, pelleted diets were the same for all animal groups (Special Diet Service, Essex, UK, product code 801010). Animals were culled at week 10 for follow-up experiments. All experiments were approved by the Ethics Committee of University C...
Experimental design
F 1 -generation offspring GF, exGF and CON mice were culled in adulthood at 10 weeks. Brains were collected for quantitative real-time PCR (qRT-PCR), for protein analysis and for transmission electron microscopy. For full detailed experimental flow with relevant animal number per experiment, see.
Transmission electron microscopy
Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK). For each specimen, semi-thin (0.5 µm) and thin (70-90 nm) sections were obtained from polymerized blocks using a Reichert-Jung Ultracut E ultramicrotome (Leica-Microsystems, Wetzlar, Germany). Semi-thin sections were stained with toluidine blue and examined using a light microscope. Thin sections from selected areas of the trimmed blocks were made and collected on formvar-coated copper grids (Agar Scientific). Thin sections were double contrasted with 2% uranyl acetate and Reynolds lead citrate stain, and examined using a Jeol 2000FXII transmission...
Measurement outputs
What raw and processed outputs should exist?
F 1 -generation offspring GF, exGF and CON mice were culled in adulthood at 10 weeks. Brains were collected for quantitative real-time PCR (qRT-PCR), for protein analysis and fo...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Total protein was extracted using a commercially available mirVana PARIS RNA and Native Protein Purification kit (Thermo Fisher Scientific). Protein levels were detected using a...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and d...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK).
from paperScoring or quantification
Quantify the primary readouts for this experiment: F 1 -generation offspring GF, exGF and CON mice were culled in adulthood at 10 weeks. Brains were collected for quantitative real-time PCR (qRT-PCR), for protein analysis and fo...; RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene...; Total protein was extracted using a commercially available mirVana PARIS RNA and Native Protein Purification kit (Thermo Fisher Scientific). Protein levels were detected using a...; Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and d....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene...; All statistics used for analysis of differential gene expression were according to the description of R package DESeq2 with default parameters. Multiple-testing corrections were...
from paperReporting output
Report representative outputs alongside summary comparisons for F 1 -generation offspring GF, exGF and CON mice were culled in adulthood at 10 weeks. Brains were collected for quantitative real-time PCR (qRT-PCR), for protein analysis and fo..., RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene..., Total protein was extracted using a commercially available mirVana PARIS RNA and Native Protein Purification kit (Thermo Fisher Scientific). Protein levels were detected using a..., Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and d....
inferred from protocolStructured statistical methods
RNA was reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) in a G-storm thermocycler (G-storm, Surrey, UK). Gene...; All statistics used for analysis of differential gene expression were according to the description of R package DESeq2 with default parameters. Multiple-testing corrections were...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
GF and CON Swiss Webster breeding pairs were obtained from Taconic (Germantown, NY, USA). GF mice were housed in gnotobiotic isolators under a strict 12-h light/dark cycle in cages of 4-5 animals. On postnatal day 21, just after weaning, exGF mice were taken from isolators and housed next to CON mice. CON and exGF mice were housed in the standard animal unit, which allows for exGF mice to be exposed to environmental microbes resulting in colonization. CON mice are similarly housed under regulated conditions (temperature 20-21 °C, 55-60% humidity), under the same 12-h light/dark cycle and housed 4-5 per cage. Autoclaved, pelleted diets were the same for all animal groups (Special Diet Service, Essex, UK, product code 801010). Animals were culled at week 10 for follow-up experiments. All experiments were approved by the Ethics Committee of University College Cork and the Irish Department of Health authorities.
F 1 -generation offspring GF, exGF and CON mice were culled in adulthood at 10 weeks. Brains were collected for quantitative real-time PCR (qRT-PCR), for protein analysis and for transmission electron microscopy. For full detailed experimental flow with relevant animal number per experiment, see.
Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK). For each specimen, semi-thin (0.5 µm) and thin (70-90 nm) sections were obtained from polymerized blocks using a Reichert-Jung Ultracut E ultramicrotome (Leica-Microsystems, Wetzlar, Germany). Semi-thin sections were stained with toluidine blue and examined using a light microscope. Thin sections from selected areas of the trimmed blocks were made and collected on formvar-coated copper grids (Agar Scientific). Thin sections were double contrasted with 2% uranyl acetate and Reynolds lead citrate stain, and examined using a Jeol 2000FXII transmission electron microscope (JEOL, Peabody, MA, USA), operated at 80 kV. Electron micrographs were obtained of areas of interest with a Megaview-III digital camera and AnalySIS software (EMSIS, Münster, Germany). A minimum of 50 myelinated axons were measured per animal.
Machine-readable layer
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