Gut Microbiota Composition in Male Rat Models under Different Nutritional Status and Physical Activity and Its Association with Serum Leptin and Ghrelin Levels methods
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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...
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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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