Limits...
A biomolecular isolation framework for eco-systems biology.

Roume H, Muller EE, Cordes T, Renaut J, Hiller K, Wilmes P - ISME J (2012)

Bottom Line: The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods.To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest.The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

View Article: PubMed Central - PubMed

Affiliation: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.

ABSTRACT
Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single samples to facilitate meaningful data integration, analysis and modeling. We have developed a new methodological framework for the reproducible isolation of high-quality genomic DNA, large and small RNA, proteins, and polar and non-polar metabolites from single unique mixed microbial community samples. The methodology is based around reproducible cryogenic sample preservation and cell lysis. Metabolites are extracted first using organic solvents, followed by the sequential isolation of nucleic acids and proteins using chromatographic spin columns. The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest. The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

Show MeSH

Related in: MedlinePlus

Quality and quantity of biomacromolecular fractions isolated from the representative LAO-enriched microbial community sample using either the NA-, QA- and TR-based method (following prior metabolite extractions) or using the reference methods (no metabolite extractions were carried out before the respective extractions). (a and b) Representative Agilent Bioanalyzer 2100 electropherograms of the total RNA and small RNA fractions, respectively. (c) Agarose gel image highlighting representative genomic DNA fractions obtained (Mean amount (n=3) loaded in μg±s.d., from right to left; NA: 0.35±0.17; QA: 0.35±0.08; TR: 0.83±0.85; RM: 0.08±0.04) and (d) SDS-PAGE image of representative protein fractions (Mean amount (n=3) loaded in μg±s.d., from right to left; NA, first elution: 3.20±0.19; QA: 5.44±1.06; TR: 3.88±0.30; RM: 4.62±0.09). The arrow and the box represent the dominant gel band which was submitted to tryptic digestion and MALDI-ToF/ToF analysis. (e) Biomacromolecular yield obtained for the  small RNA, □ total and large RNA,  DNA and  protein (first elution) fractions (n=5, error bars represent s.d.). FU, fluorescent unit; L, ladder; M, marker; NA, NA-based method; Norm., normalized; nt, nucleotides; RM, reference methods; TR, TR-based method; QA, QA-based method.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3526178&req=5

fig4: Quality and quantity of biomacromolecular fractions isolated from the representative LAO-enriched microbial community sample using either the NA-, QA- and TR-based method (following prior metabolite extractions) or using the reference methods (no metabolite extractions were carried out before the respective extractions). (a and b) Representative Agilent Bioanalyzer 2100 electropherograms of the total RNA and small RNA fractions, respectively. (c) Agarose gel image highlighting representative genomic DNA fractions obtained (Mean amount (n=3) loaded in μg±s.d., from right to left; NA: 0.35±0.17; QA: 0.35±0.08; TR: 0.83±0.85; RM: 0.08±0.04) and (d) SDS-PAGE image of representative protein fractions (Mean amount (n=3) loaded in μg±s.d., from right to left; NA, first elution: 3.20±0.19; QA: 5.44±1.06; TR: 3.88±0.30; RM: 4.62±0.09). The arrow and the box represent the dominant gel band which was submitted to tryptic digestion and MALDI-ToF/ToF analysis. (e) Biomacromolecular yield obtained for the small RNA, □ total and large RNA, DNA and protein (first elution) fractions (n=5, error bars represent s.d.). FU, fluorescent unit; L, ladder; M, marker; NA, NA-based method; Norm., normalized; nt, nucleotides; RM, reference methods; TR, TR-based method; QA, QA-based method.

Mentions: The absorbance ratios at 260/280 nm of all RNA fractions are between 1.8 and 2.1 (Supplementary Table 3), indicating that overall high-quality RNA was extracted using the different protocols. The Agilent Bioanalyzer 2100 electropherograms (Figure 4a) show distinct peaks between 100 and 4500 nt representing the total RNA fractions obtained using the different methods. The 23S/16S rRNA ratios vary depending on the RNA extraction method used from 0.7 to 1.5 (Supplementary Table 3). However, integrity of the isolated RNA was mainly assessed using the RNA integrity number (RIN) score, which is now commonly accepted as a better RNA quality indicator (Fleige and Pfaffl, 2006; Jahn et al., 2008). The RIN scores obtained with the NA-based method (7.03±1.20) are similar to those obtained using the Qiagen RNeasy Mini kit-based reference method (6.60±0.88; Kruskal–Wallis, P=0.806, n=5) and the sequential biomolecular extraction based on the TR-based method (7.44±0.28; Kruskal–Wallis, P=0.352, n=5) but lower than those obtained for the QA-based method (9.68±0.05; Kruskal–Wallis, P=0.011, n=5), which had a very high and consistent score. The RNA fraction obtained using the TR-based method is particularly enriched in small RNAs, indicative of extensive RNA degradation resulting in a non-representative RNA fraction (Figure 4a). Overall, the RNA fractions derived using the simultaneous and sequential biomolecular isolation methods are of sufficient quality for downstream ribosomal RNA removal, reverse transcription and high-throughput cDNA sequencing (He et al., 2010).


A biomolecular isolation framework for eco-systems biology.

Roume H, Muller EE, Cordes T, Renaut J, Hiller K, Wilmes P - ISME J (2012)

Quality and quantity of biomacromolecular fractions isolated from the representative LAO-enriched microbial community sample using either the NA-, QA- and TR-based method (following prior metabolite extractions) or using the reference methods (no metabolite extractions were carried out before the respective extractions). (a and b) Representative Agilent Bioanalyzer 2100 electropherograms of the total RNA and small RNA fractions, respectively. (c) Agarose gel image highlighting representative genomic DNA fractions obtained (Mean amount (n=3) loaded in μg±s.d., from right to left; NA: 0.35±0.17; QA: 0.35±0.08; TR: 0.83±0.85; RM: 0.08±0.04) and (d) SDS-PAGE image of representative protein fractions (Mean amount (n=3) loaded in μg±s.d., from right to left; NA, first elution: 3.20±0.19; QA: 5.44±1.06; TR: 3.88±0.30; RM: 4.62±0.09). The arrow and the box represent the dominant gel band which was submitted to tryptic digestion and MALDI-ToF/ToF analysis. (e) Biomacromolecular yield obtained for the  small RNA, □ total and large RNA,  DNA and  protein (first elution) fractions (n=5, error bars represent s.d.). FU, fluorescent unit; L, ladder; M, marker; NA, NA-based method; Norm., normalized; nt, nucleotides; RM, reference methods; TR, TR-based method; QA, QA-based method.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526178&req=5

fig4: Quality and quantity of biomacromolecular fractions isolated from the representative LAO-enriched microbial community sample using either the NA-, QA- and TR-based method (following prior metabolite extractions) or using the reference methods (no metabolite extractions were carried out before the respective extractions). (a and b) Representative Agilent Bioanalyzer 2100 electropherograms of the total RNA and small RNA fractions, respectively. (c) Agarose gel image highlighting representative genomic DNA fractions obtained (Mean amount (n=3) loaded in μg±s.d., from right to left; NA: 0.35±0.17; QA: 0.35±0.08; TR: 0.83±0.85; RM: 0.08±0.04) and (d) SDS-PAGE image of representative protein fractions (Mean amount (n=3) loaded in μg±s.d., from right to left; NA, first elution: 3.20±0.19; QA: 5.44±1.06; TR: 3.88±0.30; RM: 4.62±0.09). The arrow and the box represent the dominant gel band which was submitted to tryptic digestion and MALDI-ToF/ToF analysis. (e) Biomacromolecular yield obtained for the small RNA, □ total and large RNA, DNA and protein (first elution) fractions (n=5, error bars represent s.d.). FU, fluorescent unit; L, ladder; M, marker; NA, NA-based method; Norm., normalized; nt, nucleotides; RM, reference methods; TR, TR-based method; QA, QA-based method.
Mentions: The absorbance ratios at 260/280 nm of all RNA fractions are between 1.8 and 2.1 (Supplementary Table 3), indicating that overall high-quality RNA was extracted using the different protocols. The Agilent Bioanalyzer 2100 electropherograms (Figure 4a) show distinct peaks between 100 and 4500 nt representing the total RNA fractions obtained using the different methods. The 23S/16S rRNA ratios vary depending on the RNA extraction method used from 0.7 to 1.5 (Supplementary Table 3). However, integrity of the isolated RNA was mainly assessed using the RNA integrity number (RIN) score, which is now commonly accepted as a better RNA quality indicator (Fleige and Pfaffl, 2006; Jahn et al., 2008). The RIN scores obtained with the NA-based method (7.03±1.20) are similar to those obtained using the Qiagen RNeasy Mini kit-based reference method (6.60±0.88; Kruskal–Wallis, P=0.806, n=5) and the sequential biomolecular extraction based on the TR-based method (7.44±0.28; Kruskal–Wallis, P=0.352, n=5) but lower than those obtained for the QA-based method (9.68±0.05; Kruskal–Wallis, P=0.011, n=5), which had a very high and consistent score. The RNA fraction obtained using the TR-based method is particularly enriched in small RNAs, indicative of extensive RNA degradation resulting in a non-representative RNA fraction (Figure 4a). Overall, the RNA fractions derived using the simultaneous and sequential biomolecular isolation methods are of sufficient quality for downstream ribosomal RNA removal, reverse transcription and high-throughput cDNA sequencing (He et al., 2010).

Bottom Line: The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods.To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest.The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

View Article: PubMed Central - PubMed

Affiliation: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.

ABSTRACT
Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single samples to facilitate meaningful data integration, analysis and modeling. We have developed a new methodological framework for the reproducible isolation of high-quality genomic DNA, large and small RNA, proteins, and polar and non-polar metabolites from single unique mixed microbial community samples. The methodology is based around reproducible cryogenic sample preservation and cell lysis. Metabolites are extracted first using organic solvents, followed by the sequential isolation of nucleic acids and proteins using chromatographic spin columns. The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest. The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

Show MeSH
Related in: MedlinePlus