NanoDrop Microvolume Quantitation of Nucleic Acids

Desjardins, Philippe; Conklin, Deborah

doi:10.3791/2565

Cited by 301 publications

(237 citation statements)

References 2 publications

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“…Full-length gene amplicons were generated for each gene included in this study (see Table 1 for primer sequences). The amplicons were evaluated via NanoDrop (30) and diluted in 2 g/ml sheared salmon sperm DNA to 10 7 copies/l. The quantitative PCRs (qPCRs) were conducted with the Power SYBR green PCR master mix (Applied Biosystems, Foster City, CA) containing each of the funbut primers at 500 nM and 1 l of but gene amplicon DNA in 20 l on a Stratagene 3005P thermocycler (San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine

Trachsel

Bayles

Looft

et al. 2016

Appl Environ Microbiol

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Studying the host-associated butyrate-producing bacterial community is important, because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl coenzyme A (CoA):acetate-CoA transferase (EC 2.3.8.3) as a gene of primary importance for butyrate production in intestinal ecosystems; however, this gene family (but) remains poorly defined. We developed tools for the analysis of butyrate-producing bacteria based on 12 putative but genes identified in the genomes of nine butyrate-producing bacteria obtained from the swine intestinal tract. Functional analyses revealed that eight of these genes had strong But enzyme activity. When but paralogues were found within a genome, only one gene per genome encoded strong activity, with the exception of one strain in which no gene encoded strong But activity. Degenerate primers were designed to amplify the functional but genes and were tested by amplifying environmental but sequences from DNA and RNA extracted from swine colonic contents. The results show diverse but sequences from swine-associated butyrate-producing bacteria, most of which clustered near functionally confirmed sequences. Here, we describe tools and a framework that allow the bacterial butyrate-producing community to be profiled in the context of animal health and disease. IMPORTANCEButyrate is a compound produced by the microbiota in the intestinal tracts of animals. This compound is of critical importance for intestinal health, and yet studying its production by diverse intestinal bacteria is technically challenging. Here, we present an additional way to study the butyrate-producing community of bacteria using one degenerate primer set that selectively targets genes experimentally demonstrated to encode butyrate production. This work will enable researchers to more easily study this very important bacterial function that has implications for host health and resistance to disease. S hort-chain fatty acids (SCFAs) play a central role in the maintenance of colonic homeostasis, which is the delicate balance between the host, its immune system, and the gastrointestinal microbial partners (1). Butyrate in particular has potent effects on host tissues. As with other SCFAs, butyrate is consumed by the host as an energy source; however, unlike the other common SCFAs, such as propionate and acetate, butyrate is the preferred energy source for colonocytes (2) and is rapidly absorbed and used by the colonic epithelium. This rapid oxidation of butyrate reduces local oxygen concentrations, causing the epithelia to become hypoxic and thus limiting the growth of facultative aerobic pathogens, such as Salmonella species (3, 4). In addition, butyrate alters host gene expression to promote immune tolerance to the colonic microbiota and to improve the barrier function of the colonic epithelium. For example, butyrate has been shown to increase the secretion of antimicrobial peptides and mucus as well as the expression of tight junction proteins, thickening and strengthenin...

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Section: Methodsmentioning

confidence: 99%

Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine

Trachsel

Bayles

Looft

et al. 2016

Appl Environ Microbiol

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“…Total RNA was isolated from 0.5 g liver tissue using TRIZOL (Invitrogen, USA) and quantified by measuring absorbance at 260 nm in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, USA) (Desjardins and Conklin, 2011). cDNA was converted by M-MLV reverse transcriptase (Promega, USA) as indicated by the manufacturer protocol (heated to 95°C for 2 min, incubated for 5 min at 70°C, and then chilled on ice; cDNA was generated for 1 h at 37°C).…”

Section: Assessment Of Aldehyde Dehydrogenase (Aldh) Aldo-keto Reducmentioning

confidence: 99%

Relationship between liver and low rumen pH in goat

Xie¹,

Jiang²,

Ye³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. The aim of this study was to analyze the response of dry goat liver to sub-acute ruminal acidosis induced by a highly concentrated diet. Non-pregnant, non-lactating female Poll-goats (N = 12) were randomly assigned to either a high-concentrate (HG) or a low-concentrate (LG) diet. Low rumen pH was successfully induced with HG (more than 3 h with rumen pH < 5.8). The plasma lipopolysaccharide concentration was significantly decreased in the HG compared with LG group (P < 0.05). Proteomic analysis showed that aldehyde dehydrogenases and microsomal glutathione S-transferase was downregulated in the HG group, whereas aldo-keto reductase was upregulated compared in the LG group. The abundance of mRNA for these proteins were also correspondingly increased (aldehyde dehydrogenases and microsomal glutathione-S-transferase) or decreased (aldo-keto reductase) in the HG group. Malondialdehyde content in the liver was decreased in the HG group compared to the LG group. These data indicate that the expression of hepatic proteins alters the regulation of endogenous lipopolysaccharide during low rumen pH in dry dairy goats. In particular, the protective effect of the liver may occur through inhibition of aldehyde and/or peroxide formation.

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“…RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (Desjardins and Conklin, 2011). The purity of RNA (A 260 /A 280 ) for all samples was above 1.85.…”

Section: Real-time Polymerase Chain Reaction (Rt-pcr)mentioning

confidence: 99%

Effect of high-concentrate diet on amino acid transporter expression and milk quality in Holstein dairy cows

Xie

Zhang

et al. 2015

Genet. Mol. Res.

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ABSTRACT. In order to evaluate the effect of high-concentrate diet supplementation on milk protein content, six Holstein dairy cows were assigned into high-concentrate diet (HC) or low-concentrate diet (LC) groups (N = 3/group) for 50 days. With regard to milk protein, HC feeding significantly reduced the percentage of milk protein (P < 0.01), and milk protein yield also reduced. The milk somatic cell count numbers and N-acetyl-D-glucosaminidase activity was significantly higher (P < 0.01) in the HC group than in the LC group. A pre-column derivatization procedure of o-phthalaldehyde was used to analyze the milk amino acid profile, the contents of Asp, Gln, Ala, Ile, Leu, and Lys were significantly lower in milk (P < 0.05), but Arg and Phe were significantly higher (P < 0.05) in the HC group than in the LC group. The mRNA abundance for amino acid transporters SLC7A8, SLC7A10 (P < 0.05), SLC1A3 (P < 0.05), and SLC16A10 (P < 0.05) were decreased in the HC group. These data indicate that expression of amino acid transporters alters regulation of amino acid utilization and decreases milk quality in dairy cows.

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NanoDrop Microvolume Quantitation of Nucleic Acids

Cited by 301 publications

References 2 publications

Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine

Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine

Relationship between liver and low rumen pH in goat

Effect of high-concentrate diet on amino acid transporter expression and milk quality in Holstein dairy cows

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