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Automated parallel isolation of multiple species of non-coding RNAs by the reciprocal circulating chromatography method.

Miyauchi K, Ohara T, Suzuki T - Nucleic Acids Res. (2007)

Bottom Line: However, there have been no general and convenient strategies for isolation of individual RNAs.RCC employs multiple tip-columns packed with solid-phase DNA probes to isolate multiple RNA species from a common sample of total RNAs.A pilot RCC instrument successfully isolated various ncRNAs from E. coli, yeast and mouse.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, Graduate School of Frontier Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Recent genome-wide transcriptome analysis has identified diverse classes of non-coding RNAs (ncRNAs), some of which have been demonstrated to be functional, regulatory RNAs involved in various biological processes. Maturation of RNA molecules through various post-transcriptional processing events, including splicing, modification, editing and trimming of both ends, is required for correct folding and proper function of RNA molecules. To characterize post-transcriptional modifications and terminal chemical structures of fully processed native RNAs, it is necessary to isolate individual RNA species from a limited quantity and complex mixture of cellular RNAs. However, there have been no general and convenient strategies for isolation of individual RNAs. We describe here the first example of automated parallel isolation of individual ncRNAs using a novel method named 'reciprocal circulating chromatography (RCC)'. RCC employs multiple tip-columns packed with solid-phase DNA probes to isolate multiple RNA species from a common sample of total RNAs. A pilot RCC instrument successfully isolated various ncRNAs from E. coli, yeast and mouse.

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Fitting to the theoretical model for RCC. (A) Isolation profile of E. coli tRNAPro2 simulated by the recurrence equation (6) using the initial conditions C0 = 0.462 µM and Nmax = 1.755 nmol. Yields of E. coli tRNAPro2 after n pipetting cycles with various equilibrium constants (K) were plotted (solid lines). Dashed line represents a plot when K is the infinite value. Kapp = 11 µM−1 is an experimental value fitted to the theoretical model for RCC (dotted line). (B) Experimental values of E. coli tRNAPro2 concentration (Cn) in the sample reservoir were quantified by dot blot hybridization and fitted to the theoretical equation. Kapp was determined to be approximately 1.1 × 107 M−1 with the coefficient of determination (R2) 0.888. (C) Predicted yields calculated from the theoretical equation with Kapp = 1.1 × 107 M−1 were compared with actual experimental yields quantified by northern hybridization. Initial conditions are described below the graph.
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Figure 2: Fitting to the theoretical model for RCC. (A) Isolation profile of E. coli tRNAPro2 simulated by the recurrence equation (6) using the initial conditions C0 = 0.462 µM and Nmax = 1.755 nmol. Yields of E. coli tRNAPro2 after n pipetting cycles with various equilibrium constants (K) were plotted (solid lines). Dashed line represents a plot when K is the infinite value. Kapp = 11 µM−1 is an experimental value fitted to the theoretical model for RCC (dotted line). (B) Experimental values of E. coli tRNAPro2 concentration (Cn) in the sample reservoir were quantified by dot blot hybridization and fitted to the theoretical equation. Kapp was determined to be approximately 1.1 × 107 M−1 with the coefficient of determination (R2) 0.888. (C) Predicted yields calculated from the theoretical equation with Kapp = 1.1 × 107 M−1 were compared with actual experimental yields quantified by northern hybridization. Initial conditions are described below the graph.

Mentions: To validate the theoretical model of RCC, we simulated an isolation profile of E. coli tRNAPro2 from total RNA solution, using a 30 mer 3′-biotinylated DNA probe complementary to the anticodon region of tRNAPro2. The exact concentration of E. coli tRNAPro2 (C0) in a 1.6 mg/ml E. coli total RNA solution was determined to be 0.462 µM by dot blot hybridization using highly purified E. coli tRNAPro2 as a standard control. The approximate value of Nmax was determined to be 1.755 nmol from the yield of E. coli tRNAPro2 isolated from an excess (8 mg/ml) of E. coli total RNA. Using these initial conditions for the recurrence formula (Equation (6)), the yield of E. coli tRNAPro2 versus number of pipetting cycles was plotted using various equilibrium constants (K ) as shown in Figure 2A. The number of pipetting cycles at which the yield reaches a plateau varies drastically depending on the K values, suggesting that the total yield depends on the strength of the ligand–target affinity. In addition, the number of pipetting cycles sufficient for the efficient isolation of a target RNA can be predicted by this graph.Figure 2.


Automated parallel isolation of multiple species of non-coding RNAs by the reciprocal circulating chromatography method.

Miyauchi K, Ohara T, Suzuki T - Nucleic Acids Res. (2007)

Fitting to the theoretical model for RCC. (A) Isolation profile of E. coli tRNAPro2 simulated by the recurrence equation (6) using the initial conditions C0 = 0.462 µM and Nmax = 1.755 nmol. Yields of E. coli tRNAPro2 after n pipetting cycles with various equilibrium constants (K) were plotted (solid lines). Dashed line represents a plot when K is the infinite value. Kapp = 11 µM−1 is an experimental value fitted to the theoretical model for RCC (dotted line). (B) Experimental values of E. coli tRNAPro2 concentration (Cn) in the sample reservoir were quantified by dot blot hybridization and fitted to the theoretical equation. Kapp was determined to be approximately 1.1 × 107 M−1 with the coefficient of determination (R2) 0.888. (C) Predicted yields calculated from the theoretical equation with Kapp = 1.1 × 107 M−1 were compared with actual experimental yields quantified by northern hybridization. Initial conditions are described below the graph.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851638&req=5

Figure 2: Fitting to the theoretical model for RCC. (A) Isolation profile of E. coli tRNAPro2 simulated by the recurrence equation (6) using the initial conditions C0 = 0.462 µM and Nmax = 1.755 nmol. Yields of E. coli tRNAPro2 after n pipetting cycles with various equilibrium constants (K) were plotted (solid lines). Dashed line represents a plot when K is the infinite value. Kapp = 11 µM−1 is an experimental value fitted to the theoretical model for RCC (dotted line). (B) Experimental values of E. coli tRNAPro2 concentration (Cn) in the sample reservoir were quantified by dot blot hybridization and fitted to the theoretical equation. Kapp was determined to be approximately 1.1 × 107 M−1 with the coefficient of determination (R2) 0.888. (C) Predicted yields calculated from the theoretical equation with Kapp = 1.1 × 107 M−1 were compared with actual experimental yields quantified by northern hybridization. Initial conditions are described below the graph.
Mentions: To validate the theoretical model of RCC, we simulated an isolation profile of E. coli tRNAPro2 from total RNA solution, using a 30 mer 3′-biotinylated DNA probe complementary to the anticodon region of tRNAPro2. The exact concentration of E. coli tRNAPro2 (C0) in a 1.6 mg/ml E. coli total RNA solution was determined to be 0.462 µM by dot blot hybridization using highly purified E. coli tRNAPro2 as a standard control. The approximate value of Nmax was determined to be 1.755 nmol from the yield of E. coli tRNAPro2 isolated from an excess (8 mg/ml) of E. coli total RNA. Using these initial conditions for the recurrence formula (Equation (6)), the yield of E. coli tRNAPro2 versus number of pipetting cycles was plotted using various equilibrium constants (K ) as shown in Figure 2A. The number of pipetting cycles at which the yield reaches a plateau varies drastically depending on the K values, suggesting that the total yield depends on the strength of the ligand–target affinity. In addition, the number of pipetting cycles sufficient for the efficient isolation of a target RNA can be predicted by this graph.Figure 2.

Bottom Line: However, there have been no general and convenient strategies for isolation of individual RNAs.RCC employs multiple tip-columns packed with solid-phase DNA probes to isolate multiple RNA species from a common sample of total RNAs.A pilot RCC instrument successfully isolated various ncRNAs from E. coli, yeast and mouse.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, Graduate School of Frontier Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Recent genome-wide transcriptome analysis has identified diverse classes of non-coding RNAs (ncRNAs), some of which have been demonstrated to be functional, regulatory RNAs involved in various biological processes. Maturation of RNA molecules through various post-transcriptional processing events, including splicing, modification, editing and trimming of both ends, is required for correct folding and proper function of RNA molecules. To characterize post-transcriptional modifications and terminal chemical structures of fully processed native RNAs, it is necessary to isolate individual RNA species from a limited quantity and complex mixture of cellular RNAs. However, there have been no general and convenient strategies for isolation of individual RNAs. We describe here the first example of automated parallel isolation of individual ncRNAs using a novel method named 'reciprocal circulating chromatography (RCC)'. RCC employs multiple tip-columns packed with solid-phase DNA probes to isolate multiple RNA species from a common sample of total RNAs. A pilot RCC instrument successfully isolated various ncRNAs from E. coli, yeast and mouse.

Show MeSH
Related in: MedlinePlus