Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep

Béra-Maillet, Christel; Mosoni, Pascale; Kwasiborski, Anthony; Suau, Florent; Ribot, Yves; Forano, Evelyne

doi:10.1016/j.mimet.2008.11.009

Cited by 35 publications

(35 citation statements)

References 41 publications

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“…Our observation clearly demonstrates the divergence of GH 10 xylanase genes at the genomic and transcriptional levels; this divergence might be ascribed to the specific expression of genes during ruminal fermentation, which suggests that evaluation of functional genes at the RNA level is more reliable in providing an understanding of the natural course of xylanase changes. qPCR has been used to quantify gene expression (41), such as for the transcript analysis of GH genes in Phanerochaete chrysosporium (42,43) and Fibrobacter succinogenes S85 (23). The use of an appropriate reference gene is extremely important for quantitation because many factors can affect RNA extraction efficiency and/or recovery (44).…”

Section: Discussionmentioning

confidence: 99%

“…For example, xylanase activity in goat rumen peaks at 6 h postfeeding and then decreases gradually over the next 3 h (21,22). The transcriptional levels of GH genes from some microbes, such as F. succinogenes (23) and Phanerochaete chrysosporium (24), have been investigated and quantified; however, no studies have reported the transcriptional levels of GH 10 xylanase genes in the rumen after feeding.…”

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confidence: 99%

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Comparative Quantitative Analysis of Gene Expression Profiles of Glycoside Hydrolase Family 10 Xylanases in the Sheep Rumen during a Feeding Cycle

Zhao

Yang

et al. 2013

Appl Environ Microbiol

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Xylanase is a crucial hydrolytic enzyme that degrades plant polysaccharides in the rumen. To date, there is no information on the genetic composition and expression characteristics of ruminal xylanase during feeding cycles of ruminants. Here, the major xylanase of the glycoside hydrolase family 10 (GH 10) from the rumen of small-tail Han sheep was investigated during a feeding cycle. We identified 44 distinct GH 10 xylanase gene fragments at both the genomic and transcriptional levels. Comparison of their relative abundance showed that results from the evaluation of functional genes at the transcriptional level are more reliable indicators for understanding fluctuations in xylanase levels. The expression patterns of six xylanase genes, detected at all time points of the feeding cycle, were investigated; we observed a complex trend of gene expression over 24 h, revealing the dynamic expression of xylanases in the rumen. Further correlation analysis indicated that the rumen is a dynamic ecosystem where the transcript profiles of xylanase genes are closely related to ruminal conditions, especially rumen pH and bacterial population. Given the huge diversity and changing composition of enzymes over the entire rumen, this research provides valuable information for understanding the role of functional genes in the digestion of plant material.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Comparative Quantitative Analysis of Gene Expression Profiles of Glycoside Hydrolase Family 10 Xylanases in the Sheep Rumen during a Feeding Cycle

Zhao

Yang

et al. 2013

Appl Environ Microbiol

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“…To determine if our improved method was suitable for the isolation of RNA fragments with a size similar to the size of the fragments that are commonly generated in the library construction process and to the read length that is typical for sequence reads generated on NGS platforms, we designed primers targeting an internal region (ranging between 186 bp and 240 bp) of three CAZyme genes (celF, xynD, and cel3) from the fibrolytic rumen bacterium Fibrobacter succinogenes S85 [19]. The three fragments were detected by RT-PCR, suggesting that our method can be used, similar to the methods reported by Béra-Mailett et al (2009) and , to isolate ≤200 bp fragments of functional mRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…The synthesized cDNA was used to amplify fragments of three CAZyme genes (celF, xynD, and cel3) from Fibrobacter succinogenes S85 as previously described [19,21]. Subsequent PCR reactions were performed using 2x AmpliTag Gold PCR master mix (Invitrogen) with 1 µL first strand cDNA as template.…”

Section: Reverse Transcription and Pcrmentioning

confidence: 99%

“…All successful RNA extraction techniques have in common that they are generating significant amounts of intact RNA at a relatively low cost and it can be hypothesized that the more distinct RNA extraction methods are publicly available the more likely it will be that a suitable protocol will be readily available for future metatranscriptome studies. As there has been an increased interest in generating an expression profile of the rumen microbiome, several protocols for extraction of total RNA from the liquid and solid phase of the rumen have recently been developed [19][20][21]. Average total RNA yields as high as ~200 μg of total RNA/(mL rumen fluid) and ~110 μg of total RNA/(g solid digesta) had been reported from a cannulated Holstein cow and a cannulated muskoxen respectively [20,21].…”

Section: Introductionmentioning

confidence: 99%

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Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics

Piao¹,

Markillie²,

Culley³

et al. 2013

AiM

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Metatranscriptomics-gene express profiling via DNA sequencing-is a powerful tool to identify genes that are actively expressed and might contribute to the phenotype of individual organisms or the phenome (the sum of several phenotypes) of a microbial community. Furthermore, metatranscriptome studies can result in extensive catalogues of genes that encode for enzymes of industrial relevance. In both cases, a major challenge for generating a high quality metatranscriptome is the extreme lability of RNA and its susceptibility to ubiquitous RNAses. The microbial community (the microbiome) of the cow rumen efficiently degrades lignocelullosic biomass, generates significant amounts of methane, a greenhouse gas twenty times more potent than carbon dioxide, and is of general importance for the physiological wellbeing of the host animal. Metatranscriptomes of the rumen microbiome from animals kept under different conditions and from various types of rumen-incubated biomass can be expected to provide new insights into these highly interesting phenotypes and subsequently provide the framework for an enhanced understanding of this socioeconomically important ecosystem. The ability to isolate large amounts of intact RNA will significantly facilitate accurate transcript annotation and expression profiling. Here we report a method that combines mechanical disruption with chemical homogenization of the sample material and consistently yields 1 mg of intact RNA from 1 g of rumen-incubated biofuel feedstock. The yield of total RNA obtained with our method exceeds the RNA yield achieved with previously reported isolation techniques, which renders RNA isolated with the method presented here as an ideal starting material for metatranscriptomic analyses and other molecular biology applications that require significant amounts of starting material.

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Current Advancements in Recombinant Technology for Industrial Production of Cellulases: Part-II

et al. 2019

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Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep

Cited by 35 publications

References 41 publications

Comparative Quantitative Analysis of Gene Expression Profiles of Glycoside Hydrolase Family 10 Xylanases in the Sheep Rumen during a Feeding Cycle

Comparative Quantitative Analysis of Gene Expression Profiles of Glycoside Hydrolase Family 10 Xylanases in the Sheep Rumen during a Feeding Cycle

Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics

Current Advancements in Recombinant Technology for Industrial Production of Cellulases: Part-II

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