Cultural analyses of the human fecal donor solutions and of the murine fecal samples were performed as described previously [15 (link), 22 (link)]. For molecular analysis of the gut microbiota composition additionally assessing fastidious and uncultivable bacteria, DNA from fecal and colonic luminal samples was extracted as described previously [22–24 (link)]. In brief, DNA extracts and plasmids were quantified using Quant-iT PicoGreen reagent (Invitrogen, Paisley, UK) and adjusted to 1 ng per μl. Then, abundance of the main bacterial groups of the gut microbiota was assessed by the quantitative real time polymerase chain reaction (qRT-PCR) with group-specific 16S rRNA gene primers (Tib MolBiol, Berlin, Germany) as described previously [22–24 (link)]. The number of 16S rRNA gene copies/μg DNA of each sample was determined and frequencies of respective bacterial groups calculated proportionally to the eubacterial (V3) amplicon.
Quant it picogreen reagent
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The Quant-iT PicoGreen reagent is a fluorescent nucleic acid stain used for the quantitation of double-stranded DNA (dsDNA) in solution. It provides a sensitive and accurate method for measuring dsDNA concentrations.
Automatically generated - may contain errors
Lab products found in correlation
16s rrna gene primers, by TIB Molbiol (22 mentions)
Group specific 16s rrna gene primers, by TIB Molbiol (4 mentions)
Calf thymus dna, by Thermo Fisher Scientific (2 mentions)
G spin columns, by iNtRON Biotechnology (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Fluostar omega multi mode plate reader, by BMG Labtech (2 mentions)
Light cycler data analysis software, by Roche (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Qiaamp dna blood maxi kit, by Qiagen (1 mentions)
Flexigene dna kit, by Qiagen (1 mentions)
L ascorbic acid phosphate, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
6 well tissue culture plate, by Corning (1 mentions)
A1 hd, by Nikon (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Innuprep stool dna kit, by Analytik Jena (1 mentions)
Infinium hd methylation assay, by Illumina (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
54 protocols using quant it picogreen reagent
1
Gut Microbiota Composition Analysis
2
Quantitative Analysis of Murine Gut Microbiota
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In brief, DNA was further quantified using Quant-iT PicoGreen reagent (Invitrogen, UK) and adjusted to 1 ng/µl. Then, the main bacterial groups abundant in the murine intestinal microbiota including enterobacteria, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella species, Clostridium coccoides group, Clostridium leptum group, Mouse intestinal Bacteroides (MIB), and total eubacterial loads were determined by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or group-specific 16S rRNA gene primers (TibMolBiol, Germany) as reported previously, and numbers of 16S rRNA gene copies per nanogram DNA of each sample were assessed (Escher et al. 2018 (link)).
3
Quantifying Gut Microbiome Composition
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Total DNA from fecal samples was extracted as described previously [11] (link). Briefly, DNA was quantified using Quant-iT PicoGreen reagent (Invitrogen, UK) and adjusted to 1 ng per µL. Then, main bacterial groups abundant in the murine conventional intestinal microbiota were detected by quantitative real-time (RT) -PCR with primers specific for sequences in the 16S rRNA genes of individual bacterial species, genera or groups (Tib MolBiol, Germany) as described previously [47] (link), [48] (link), [52] (link). Numbers of 16S rRNA gene copies/ng DNA of each sample were determined and frequencies of respective bacterial groups calculated proportionally to the eubacterial (V3) amplicon.
4
Quantitative Assessment of Intestinal Microbiota
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For quantitative assessment of fastidious and even uncultivable bacteria within bacterial suspensions and fecal samples, we applied culture-independent, 16S rRNA-based methods. The total genomic DNA was extracted from respective samples and adjusted to 1 ng per µL (Quant-iT PicoGreen reagent, Invitrogen, Carlsbad, CA, USA) as reported earlier [30 (link)]. The total eubacterial loads and the main bacterial groups abundant in the murine intestinal microbiota, including gamma-Proteobacteria/Enterobacteriaceae, Enterococcus genus, Lactobacillus group, Bifidobacterium genus, Bacteroides group, including Prevotella and Porphyromonas, Clostridium coccoides group, and Clostridium leptum group, were then assessed by quantitative RT–PCR (qRT–PCR) with species-, genera- or group-specific 16S rRNA gene primers (Tib MolBiol, Berlin, Germany), as shown in Table 1 and expressed as 16S rRNA gene copies per ng DNA [34 (link)].
5
Quantification of Gut Bacterial Groups
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Human fecal donor suspensions as well as fresh ileal and colonic luminal samples were immediately transferred to liquid nitrogen and stored at −80°C until further analyses. Fecal DNA extraction was performed as reported earlier (15 (link)). Briefly, the amount of DNA was assessed with a Quant-iT PicoGreen reagent (Invitrogen, UK) and adjusted to 1 ng per μL. The main human gut bacterial groups including enterobacteria, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella species, Clostridium coccoides group, and Clostridium leptum group as well as the total eubacterial loads were determined applying quantitative real-time polymerase chain reaction (qRT-PCR) and species-, genera- or group-specific 16S rRNA gene primers (Tib MolBiol, Germany) as indicated (Figure S2 ) and further described previously (19 (link), 23 (link)) (expressed as 16S rRNA gene copies per ng DNA).
6
Quantification of Intestinal Microbiota
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DNA was extracted from fecal samples or fecal donor suspensions as described previously [27 (link), 31 (link)]. In brief, DNA was quantified by using Quant-iT PicoGreen reagent (Invitrogen, UK) and adjusted to 1 ng per μL. Then, the total eubacterial loads, as well as the main bacterial groups abundant in the murine and human intestinal microbiota including enterobacteria, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella species, Clostridium coccoides group, Clostridium leptum group, and Mouse Intestnal Bacteroides, were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genus-, or group-specific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [10 (link), 30 (link), 32 (link)] and numbers of 16S rRNA gene copies per ng DNA of each sample determined.
7
Quantification of DNA in Lyophilized Scaffolds
8
Quantifying Intestinal Microbiota Composition
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DNA was extracted from the human donor suspension and intestinal luminal content as described previously [9 (link)]. In brief, DNA was quantified by using Quant-iT PicoGreen reagent (Invitrogen, UK) and adjusted to 1 ng per μL. Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera- or group-specific 16S rRNA gene primers (Tib MolBiol, Germany) and numbers of 16S rRNA gene copies per ng DNA of each sample determined as described previously [14 (link), 15 (link), 21 (link), 22 (link)].
9
Intestinal Cytokine and Microbiome Analysis
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Expression levels of pro- and anti-inflammatory cytokines including IFN-γ, IL-22, IL-17A, and IL-10 mRNA were determined in snap frozen ileal and colonic ex vivo biopsies using Light Cycler Data Analysis Software (Roche) as stated elsewhere (30 (link)). The mRNA of the housekeeping gene for hypoxanthine-phosphoribosyltransferase was used as reference; the mRNA expression levels of the individual genes were normalized to the lowest measured value and expressed as fold expression (arbitrary units) (31 (link)).
For molecular analysis of the intestinal microbiota, DNA was extracted from fecal samples as described previously (24 (link)). Briefly, DNA extracts and plasmids were quantified using Quant-iT PicoGreen reagent (Invitrogen, Paisley, UK) and adjusted to 1 ng/µl. Then, abundance of the main bacterial groups of murine intestinal microbiota was assessed by quantitative real time-PCR with group-specific 16S rRNA gene primers (Tib MolBiol, Berlin, Germany) as described previously (5 (link), 32 (link), 33 (link)). The number of 16S rRNA gene copies per microgram DNA of each sample was determined, and frequencies of respective bacterial groups calculated proportionally to the eubacterial (V3) amplicon.
For molecular analysis of the intestinal microbiota, DNA was extracted from fecal samples as described previously (24 (link)). Briefly, DNA extracts and plasmids were quantified using Quant-iT PicoGreen reagent (Invitrogen, Paisley, UK) and adjusted to 1 ng/µl. Then, abundance of the main bacterial groups of murine intestinal microbiota was assessed by quantitative real time-PCR with group-specific 16S rRNA gene primers (Tib MolBiol, Berlin, Germany) as described previously (5 (link), 32 (link), 33 (link)). The number of 16S rRNA gene copies per microgram DNA of each sample was determined, and frequencies of respective bacterial groups calculated proportionally to the eubacterial (V3) amplicon.
10
Quantify Murine Gut Microbiota by qRT-PCR
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Major bacterial groups present in the murine gut microbiota were quantified by assessing the quantity of 16S rRNA gene copies per ng DNA applying quantitative real-time polymerase chain reaction with species-, genera- or group-specific 16S rRNA gene primers (Tib MolBiol, Berlin, Germany) (Supplementary Table S1 ).
Briefly, fecal samples were collected and preserved for further analysis with nitrogen. For DNA extraction the samples were suspended in PBS and centrifuged (16.000× g/10 min/4 °C). The probes were resuspended in 0.5 mL lysis buffer (500 mM Tris (pH 9.0), 20 mM EDTA, 10 mM NaCl, 1% SDS) and incubated with proteinase K (2 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 56 °C. After bead beating, total DNA was isolated by phenol extraction and quantified by using Quant-iT PicoGreen reagent (InvitrogenTM, Carlsbad, CA, USA) and adjusted to 1 ng per µL [6 (link),7 (link)].
Briefly, fecal samples were collected and preserved for further analysis with nitrogen. For DNA extraction the samples were suspended in PBS and centrifuged (16.000× g/10 min/4 °C). The probes were resuspended in 0.5 mL lysis buffer (500 mM Tris (pH 9.0), 20 mM EDTA, 10 mM NaCl, 1% SDS) and incubated with proteinase K (2 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 56 °C. After bead beating, total DNA was isolated by phenol extraction and quantified by using Quant-iT PicoGreen reagent (InvitrogenTM, Carlsbad, CA, USA) and adjusted to 1 ng per µL [6 (link),7 (link)].
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