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

Piao, Hailan; Markillie, Lye Meng; Culley, David E.; Mackie, Roderick I.; Hess, Matthias

doi:10.4236/aim.2013.31016

Cited by 4 publications

(3 citation statements)

References 28 publications

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“…However, obtaining good sequencing coverage for an accurate representation of the transcriptome is impeded by the difficulties of isolating total RNA with good yield, high integrity and minimal genomic DNA contamination. Owing to the short half lives and sensitive nature of bacterial RNAs, much care has to be taken to extract high quality RNA with minimal degradation that are suitable for sequencing [ 11 – 13 ]. In recent years, many commercial total RNA extraction kits employ spin-column technology and organic solvents to overcome those challenges [ 14 – 19 ].…”

Section: Introductionmentioning

confidence: 99%

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis

et al. 2015

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BackgroundNext-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform.ResultsTRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique.ConclusionsAmresco’s Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1726-3) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis

et al. 2015

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“…Poly(A) mRNA, representing transcripts of eukaryotic origin was isolated from total RNA as described previously [ 48 ]. Cleaned reads were combined and the metatransciptome was assembled using MEGAHIT [ 49 ] v.0.2.0.…”

Section: Methodsmentioning

confidence: 99%

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

et al. 2020

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The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

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“…Total RNA was isolated as described previously 73 . First, frozen rumen-incubated biomass (switchgrass) was manually ground to powder in the presence of liquid nitrogen and immediately added to TRIzol reagent (Invitrogen, Carlsbad, CA).…”

Section: Metatranscriptomics -Total Rna Extraction and Poly(a) Mrna Pmentioning

confidence: 99%

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

Hagen

Brooke

Shaw

et al. 2020

Preprint

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The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 hours in nylon bags within the rumen of cannulated dairy cows. Across a gene catalogue covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of polysaccharides such as hemicellulose, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo-and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8 and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryoteselected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggests that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

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

Cited by 4 publications

References 28 publications

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

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