Gut Microbiota Composition in Male Rat Models under Different Nutritional Status and Physical Activity and Its Association with Serum Leptin and Ghrelin Levels methods
Aim. Evidence-backed execution summary for Gut Microbiota Composition in Male Rat Models under Different Nutritional Status and Physical Activity and Its Association with Serum Leptin and Ghrelin Levels methods from Gut Microbiota Composition in Male Rat Models under Different Nutritional Status and Physical Activity and Its Association with Serum Leptin and Ghrelin Levels.
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This experiment, in seven questions
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What do I need before I start?
rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Objective
reagent used in the protocol.
- Use
- To evaluate the differences in the composition of gut microbiota in rat models under different nutritional status and physical activity and to identify their associations with serum leptin and ghrelin levels.
Hormone determination
reagent used in the protocol.
- Use
- Blood samples collected at the end of the study after the rats were sacrificed were immediately centrifuged and total ghrelin and leptin levels were determined in serum by a double antibody RIA using kits provided by Linco Research (St Charles, MI) as previously described,. The limits of sensitivity of the assays...
DNA extraction from fecal samples
reagent used in the protocol.
- Use
- Fecal samples were immediately kept after collection at -80°C and stored until analyzed. DNA extraction from 200 mg of stools was performed using the QIAamp DNA stool Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The DNA concentration was determined by absorbance at 2...
Analysis of fecal microbiota by PCR-DGGE
reagent used in the protocol.
- Use
- After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection System instrument (Bio-Rad). 6% polyacrylamide gels were prepared and electrophoresed with 1 × TAE buffer prepared from 50 × TAE buf...
Sequencing of selected bands from DGGE gels
reagent used in the protocol.
- Use
- Bands of specific interest were excised from DGGE gels with a sterile razor, placed in 40 µl sterile water and incubated at 4°C for diffusion of DNA into the water. DNA were used in a second PCR with HDA1/2 primers without GC-clamp (initial denaturation 95° for 20 s, followed 45 cycles including denat...
Microbial quantification by real-time PCR
reagent used in the protocol.
- Use
- Specific primers targeting different bacterial genera were used to characterize the fecal microbiota by real-time qPCR ( ) -. Briefly, real-time qPCR experiments were performed with a LightCycler 2.0 PCR sequence detection system using the FastStart DNA Master SYBR Green kit (Roche Diagnostics, Indianapolis,...
Microbial quantification by real-time PCR
reagent used in the protocol.
- Use
- The bacterial concentration from each fecal sample was calculated by comparing the C t values obtained from the standard curves with the LightCycler 4.0 software. Standard curves were constructed for each experiment using serial tenfold dilutions of bacterial genomic DNA (of known concentration) from pure cultures,...
Statistical analysis
reagent used in the protocol.
- Use
- Results are expressed as mean values and standard deviations. The statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL). The 16S rRNA gene copies values were converted into logarithmic values before the statistical analysis. The Kruskal-Wallis test was used to check changes in bacterial...
Animal models
Forty Male Sprague Dawley rats (160 g/5 weeks old) (Charles River Breeding Laboratory, Raleigh, NC) from the same bred were housed in a temperature-controlled room with a 12-h light/12-h dark cycles with free access to standard chow diet and water. A case-control study was performed. After 5 days acclimatization wei...
- Use
- Forty Male Sprague Dawley rats (160 g/5 weeks old) (Charles River Breeding Laboratory, Raleigh, NC) from the same bred were housed in a temperature-controlled room with a 12-h light/12-h dark cycles with free access to standard chow diet and water. A case-control study was performed. After 5 days acclimatization wei...
Analysis of fecal microbiota by PCR-DGGE
Fecal samples from each subject were examined by determining PCR-DGGE profiles as recently published by us. The V2-V3 regions of the 16S rRNA genes (positions 339 to 539 in the Escherichia coli gene) of bacteria in the fecal samples were amplified by primers HDA1-GC (5′- CGC CCG CCG CGC GCG GCG GGC GGG...
- Use
- Fecal samples from each subject were examined by determining PCR-DGGE profiles as recently published by us. The V2-V3 regions of the 16S rRNA genes (positions 339 to 539 in the Escherichia coli gene) of bacteria in the fecal samples were amplified by primers HDA1-GC (5′- CGC CCG CCG CGC GCG GCG GGC GGG...
Analysis of fecal microbiota by PCR-DGGE
After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection System instrument (Bio-Rad). 6% polyacrylamide gels were prepared and electrophoresed with 1 × TAE buffer prepared from 50 × TAE buf...
- Use
- After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection System instrument (Bio-Rad). 6% polyacrylamide gels were prepared and electrophoresed with 1 × TAE buffer prepared from 50 × TAE buf...
Sequencing of selected bands from DGGE gels
Bands of specific interest were excised from DGGE gels with a sterile razor, placed in 40 µl sterile water and incubated at 4°C for diffusion of DNA into the water. DNA were used in a second PCR with HDA1/2 primers without GC-clamp (initial denaturation 95° for 20 s, followed 45 cycles including denat...
- Use
- Bands of specific interest were excised from DGGE gels with a sterile razor, placed in 40 µl sterile water and incubated at 4°C for diffusion of DNA into the water. DNA were used in a second PCR with HDA1/2 primers without GC-clamp (initial denaturation 95° for 20 s, followed 45 cycles including denat...
Microbial quantification by real-time PCR
Specific primers targeting different bacterial genera were used to characterize the fecal microbiota by real-time qPCR ( ) -. Briefly, real-time qPCR experiments were performed with a LightCycler 2.0 PCR sequence detection system using the FastStart DNA Master SYBR Green kit (Roche Diagnostics, Indianapolis,...
- Use
- Specific primers targeting different bacterial genera were used to characterize the fecal microbiota by real-time qPCR ( ) -. Briefly, real-time qPCR experiments were performed with a LightCycler 2.0 PCR sequence detection system using the FastStart DNA Master SYBR Green kit (Roche Diagnostics, Indianapolis,...
Microbial quantification by real-time PCR
The bacterial concentration from each fecal sample was calculated by comparing the C t values obtained from the standard curves with the LightCycler 4.0 software. Standard curves were constructed for each experiment using serial tenfold dilutions of bacterial genomic DNA (of known concentration) from pure cultures,...
- Use
- The bacterial concentration from each fecal sample was calculated by comparing the C t values obtained from the standard curves with the LightCycler 4.0 software. Standard curves were constructed for each experiment using serial tenfold dilutions of bacterial genomic DNA (of known concentration) from pure cultures,...
Statistical analysis
Results are expressed as mean values and standard deviations. The statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL). The 16S rRNA gene copies values were converted into logarithmic values before the statistical analysis. The Kruskal-Wallis test was used to check changes in bacterial...
- Use
- Results are expressed as mean values and standard deviations. The statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL). The 16S rRNA gene copies values were converted into logarithmic values before the statistical analysis. The Kruskal-Wallis test was used to check changes in bacterial...
PCR-DGGE fingerprint analysis and bacterial band identification in the fecal samples
All the bands from all rat profiles in the different groups were cloned and sequenced to identify the dominant microbiota and the sequence similarity matches for bands were analyzed by MicroSeqID v2.1.1 software. Bacterial identification showed that the majority of bacteria represented in our fingerprints correspond...
- Use
- All the bands from all rat profiles in the different groups were cloned and sequenced to identify the dominant microbiota and the sequence similarity matches for bands were analyzed by MicroSeqID v2.1.1 software. Bacterial identification showed that the majority of bacteria represented in our fingerprints correspond...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Results are expressed as mean values and standard deviations. The statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL). The 16S rRNA gene copies values were converted into logarithmic values before the statistical analysis. The Kruskal-Wallis test was used to check changes in bacterial...
PCR-DGGE fingerprint analysis and bacterial band identification in the fecal samples
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All the bands from all rat profiles in the different groups were cloned and sequenced to identify the dominant microbiota and the sequence similarity matches for bands were analyzed by MicroSeqID v2.1.1 software. Bacterial identification showed that the majority of bacteria represented in our fingerprints correspond...
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Open the source paper before finalizing run-specific details.
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Open quote workflowStep-by-step procedure
What do I do, in order?
Animal models
Forty Male Sprague Dawley rats (160 g/5 weeks old) (Charles River Breeding Laboratory, Raleigh, NC) from the same bred were housed in a temperature-controlled room with a 12-h light/12-h dark cycles with free access to standard chow diet and water. A case-control study was performed. After 5 days acclimatization weight-matched animals were randomly assigned to one of four experimental groups (10 rats by group): (a) Activity based anorexia (ABA) group: rats starved by restricting food access to 23 hours per day and confined to running wheels except during a 60 min meal per day, (b) Control ABA group: rats submitted to the same food restriction schedule as ABA with no wheel access exercise, (c) Exercise group: rats feed ad libitum with free access to the activity wheel and (d) Ad libitum group: rats feed ad libitum but without access to the activity wheel. The ABA group was performed fo...
DNA extraction from fecal samples
Fecal samples were immediately kept after collection at -80°C and stored until analyzed. DNA extraction from 200 mg of stools was performed using the QIAamp DNA stool Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The DNA concentration was determined by absorbance at 260 nm (A260), and the purity was estimated by determining the A260/A280 ratio with a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE).
Analysis of fecal microbiota by PCR-DGGE
Fecal samples from each subject were examined by determining PCR-DGGE profiles as recently published by us. The V2-V3 regions of the 16S rRNA genes (positions 339 to 539 in the Escherichia coli gene) of bacteria in the fecal samples were amplified by primers HDA1-GC (5′- CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G CC TAC GGG AGG CAG CAG T-3′; (the GC clamp is in boldface) and HDA2 (5′-GTA TTA CCG CGG CTG CTG GCA C-3′) generating a 200 bp product. Aliquots (2 µL) of DNA were amplified by real-time PCR (20 µL final volume) in a 7500 Fast Real-Time PCR Systems instrument using Fast SYBR Green Master Mix and 200 nM of each of the universal primers HDA1-GC/HDA2 with the following amplification program: initial denaturation was at 95° for 20 s, amplification was carried out using 45 cycles including denaturation at 95°C for 3 s, a...
Analysis of fecal microbiota by PCR-DGGE
After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection System instrument (Bio-Rad). 6% polyacrylamide gels were prepared and electrophoresed with 1 × TAE buffer prepared from 50 × TAE buffer (2 M Tris base, 1 M glacial acetic acid, 50 mM EDTA). The denaturing gradient was formed by using two 6% acrylamide (acrylamide/bisacrylamide ratio 37.5∶1) stock solutions (Bio-Rad). The gels contained a 20-80% gradient of urea and formamide that increase in the direction of electrophoresis. Electrophoretic runs were in a Tris-acetate-EDTA buffer (TAE 1x) (40 mmol/L Tris, 20 mmol/L acetic acid, and 1 mmol/L EDTA, pH 7.4) at 130 V and 60°C for 4.5 h. Electrophoresis was stopped when a xylene cyanol dye marker reached the bottom of a gel. Gels were stained with...
Microbial quantification by real-time PCR
Specific primers targeting different bacterial genera were used to characterize the fecal microbiota by real-time qPCR ( ) -. Briefly, real-time qPCR experiments were performed with a LightCycler 2.0 PCR sequence detection system using the FastStart DNA Master SYBR Green kit (Roche Diagnostics, Indianapolis, IN). All PCR tests were carried out in duplicate with a final volume of 20 µL containing 1 µL of each fecal DNA preparation and 200 nM of each primer ( ). The thermal cycling conditions used were as follows: an initial DNA denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, primer annealing at optimal temperature ( ) for 20 s, extension at 72°C for 15 s. Finally, melt curve analysis was performed by slowly cooling the PCRs from 95 to 60°C (0.05°C per cycle) with simultaneous measurement of the SYBR...
Measurement outputs
What raw and processed outputs should exist?
In a case control study, forty male rats were randomly assigned to one of these four experimental groups: ABA group with food restriction and free access to exercise; control AB...
- 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
Fecal samples from each subject were examined by determining PCR-DGGE profiles as recently published by us. The V2-V3 regions of the 16S rRNA genes (positions 339 to 539...
- 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
After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection...
- 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
Bands of specific interest were excised from DGGE gels with a sterile razor, placed in 40 µl sterile water and incubated at 4°C for diffusion of DNA into the water. DN...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
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
Results are expressed as mean values and standard deviations.
from paperScoring or quantification
Quantify the primary readouts for this experiment: In a case control study, forty male rats were randomly assigned to one of these four experimental groups: ABA group with food restriction and free access to exercise; control AB...; Fecal samples from each subject were examined by determining PCR-DGGE profiles as recently published by us. The V2-V3 regions of the 16S rRNA genes (positions 339 to 539...; After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection...; Bands of specific interest were excised from DGGE gels with a sterile razor, placed in 40 µl sterile water and incubated at 4°C for diffusion of DNA into the water. DN....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Results are expressed as mean values and standard deviations. The statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL). The 16S rRNA gene copies v...; Variations were found in the presence or absence (qualitative) and intensity (quantitative) of the bands between rat groups in the generated host-specific fingerprints. DGGE ban...; All the bands from all rat profiles in the different groups were cloned and sequenced to identify the dominant microbiota and the sequence similarity matches for bands were anal...; Values in a row with different superscript letters are significantly different P <0.05.
from paperReporting output
Report representative outputs alongside summary comparisons for In a case control study, forty male rats were randomly assigned to one of these four experimental groups: ABA group with food restriction and free access to exercise; control AB..., Fecal samples from each subject were examined by determining PCR-DGGE profiles as recently published by us. The V2-V3 regions of the 16S rRNA genes (positions 339 to 539..., After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection..., Bands of specific interest were excised from DGGE gels with a sterile razor, placed in 40 µl sterile water and incubated at 4°C for diffusion of DNA into the water. DN....
inferred from protocolStructured statistical methods
Results are expressed as mean values and standard deviations. The statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL). The 16S rRNA gene copies v...; Variations were found in the presence or absence (qualitative) and intensity (quantitative) of the bands between rat groups in the generated host-specific fingerprints. DGGE ban...; All the bands from all rat profiles in the different groups were cloned and sequenced to identify the dominant microbiota and the sequence similarity matches for bands were anal...; Values in a row with different superscript letters are significantly different P <0.05.
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Forty Male Sprague Dawley rats (160 g/5 weeks old) (Charles River Breeding Laboratory, Raleigh, NC) from the same bred were housed in a temperature-controlled room with a 12-h light/12-h dark cycles with free access to standard chow diet and water. A case-control study was performed. After 5 days acclimatization weight-matched animals were randomly assigned to one of four experimental groups (10 rats by group): (a) Activity based anorexia (ABA) group: rats starved by restricting food access to 23 hours per day and confined to running wheels except during a 60 min meal per day, (b) Control ABA group: rats submitted to the same food restriction schedule as ABA with no wheel access exercise, (c) Exercise group: rats feed ad libitum with free access to the activity wheel and (d) Ad libitum group: rats feed ad libitum but without access to the activity wheel. The ABA group was performed following previously established model by Routtenberg and Kuznesof. For ethical reasons, ABA rats were not allowed to lose more than 20-30% of their initial body weight. All animals were housed individually in custom-designed, stainless steel cages, which in the running groups were connected to...
Fecal samples were immediately kept after collection at -80°C and stored until analyzed. DNA extraction from 200 mg of stools was performed using the QIAamp DNA stool Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The DNA concentration was determined by absorbance at 260 nm (A260), and the purity was estimated by determining the A260/A280 ratio with a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE).
Fecal samples from each subject were examined by determining PCR-DGGE profiles as recently published by us. The V2-V3 regions of the 16S rRNA genes (positions 339 to 539 in the Escherichia coli gene) of bacteria in the fecal samples were amplified by primers HDA1-GC (5′- CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G CC TAC GGG AGG CAG CAG T-3′; (the GC clamp is in boldface) and HDA2 (5′-GTA TTA CCG CGG CTG CTG GCA C-3′) generating a 200 bp product. Aliquots (2 µL) of DNA were amplified by real-time PCR (20 µL final volume) in a 7500 Fast Real-Time PCR Systems instrument using Fast SYBR Green Master Mix and 200 nM of each of the universal primers HDA1-GC/HDA2 with the following amplification program: initial denaturation was at 95° for 20 s, amplification was carried out using 45 cycles including denaturation at 95°C for 3 s, annealing at 55°C for 30 s and extension at 72°C for 1 min.
After real time PCR 15 µL of products were mixed with 6 µL loading dye before loading. Electrophoresis was performed with a DCode ™ Universal Mutation Detection System instrument (Bio-Rad). 6% polyacrylamide gels were prepared and electrophoresed with 1 × TAE buffer prepared from 50 × TAE buffer (2 M Tris base, 1 M glacial acetic acid, 50 mM EDTA). The denaturing gradient was formed by using two 6% acrylamide (acrylamide/bisacrylamide ratio 37.5∶1) stock solutions (Bio-Rad). The gels contained a 20-80% gradient of urea and formamide that increase in the direction of electrophoresis. Electrophoretic runs were in a Tris-acetate-EDTA buffer (TAE 1x) (40 mmol/L Tris, 20 mmol/L acetic acid, and 1 mmol/L EDTA, pH 7.4) at 130 V and 60°C for 4.5 h. Electrophoresis was stopped when a xylene cyanol dye marker reached the bottom of a gel. Gels were stained with ethidium bromide (0.5 mg/L) for 5 min, rinsed with deionized water, viewed by UV transillumination and photographed with Gelcapture image acquisition software (DNR Bio-Imaging Systems Ltd). All the samples were analyzed on the same DGGE run to avoid the possible influence of variations in electroph...
Specific primers targeting different bacterial genera were used to characterize the fecal microbiota by real-time qPCR ( ) -. Briefly, real-time qPCR experiments were performed with a LightCycler 2.0 PCR sequence detection system using the FastStart DNA Master SYBR Green kit (Roche Diagnostics, Indianapolis, IN). All PCR tests were carried out in duplicate with a final volume of 20 µL containing 1 µL of each fecal DNA preparation and 200 nM of each primer ( ). The thermal cycling conditions used were as follows: an initial DNA denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, primer annealing at optimal temperature ( ) for 20 s, extension at 72°C for 15 s. Finally, melt curve analysis was performed by slowly cooling the PCRs from 95 to 60°C (0.05°C per cycle) with simultaneous measurement of the SYBR Green I signal intensity. Melting-point-determination analysis allowed the confirmation of the specificity of the amplification products.
Machine-readable layer
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"text": "Forty Male Sprague Dawley rats (160 g/5 weeks old) (Charles River Breeding Laboratory, Raleigh, NC) from the same bred were housed in a temperature-controlled room with a 12-h light/12-h dark cycles with free access to standard chow diet and water. A case-control study was performed. After 5 days acclimatization weight-matched animals were randomly assigned to one of four experimental groups (10 rats by group): (a) Activity based anorexia (ABA) group: rats starved by restricting food access to 23 hours per day and confined to running wheels except during a 60 min meal per day, (b) Control ABA group: rats submitted to the same food restriction schedule as ABA with no wheel access exercise, (c) Exercise group: rats feed ad libitum with free access to the activity wheel and (d) Ad libitum group: rats feed ad libitum but without access to the activity wheel. The ABA group was performed fo..."
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