Limits...
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.

Show MeSH

Related in: MedlinePlus

Automated parallel isolation of all species of E. coli tRNA and four sRNAs. (A) Polyacrylamide gel electrophoresis of E. coli tRNAs, 4.5S and 6S RNAs isolated by the RCC instrument. RNAs were visualized by ethidium bromide staining. Several tRNAs migrated as doublet bands, which were conformers of the same tRNAs. Species of tRNAs are shown as single letter abbreviations for the corresponding amino acid and a number extension in the case of multiple tRNAs for the same amino acid. E. coli total RNA (asterisk) was used as a marker. (B) Northern blot analysis of DsrA and SraH sRNA. Strong signals were observed in the eluted fraction (elution), weak signals were visible in the starting sample (load), and no signals were found in the flow through fraction (FT). Ethidium bromide (EtBr) staining is also shown. Most of the target RNAs were highly concentrated in the eluted fractions.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851638&req=5

Figure 4: Automated parallel isolation of all species of E. coli tRNA and four sRNAs. (A) Polyacrylamide gel electrophoresis of E. coli tRNAs, 4.5S and 6S RNAs isolated by the RCC instrument. RNAs were visualized by ethidium bromide staining. Several tRNAs migrated as doublet bands, which were conformers of the same tRNAs. Species of tRNAs are shown as single letter abbreviations for the corresponding amino acid and a number extension in the case of multiple tRNAs for the same amino acid. E. coli total RNA (asterisk) was used as a marker. (B) Northern blot analysis of DsrA and SraH sRNA. Strong signals were observed in the eluted fraction (elution), weak signals were visible in the starting sample (load), and no signals were found in the flow through fraction (FT). Ethidium bromide (EtBr) staining is also shown. Most of the target RNAs were highly concentrated in the eluted fractions.

Mentions: Using a 16 mg sample of total RNA in a common reservoir as the starting material, 6 runs were conducted in which 8 different tip-columns were attached to the head of the RCC instrument for purification of the ncRNAs. Pipetting with the RNA solution in the reciprocal circulation was repeated 40 times at 66°C. Then, columns were sequentially washed and eluted by pipetting three times in 12 sets of washing tubes and 6 sets of eluting tubes at 40 and 68°C, respectively. Sufficient washing was confirmed by measuring the optical densities at 260 nm of washed fractions (not over 0.01 A260 in last tubes). Isolated RNAs collected from the eluting tubes were electrophoresed in 10% denaturing polyacrylamide gels (Figure 4A). All tRNA species were visible on the gel as a single band or as major bands with some contamination. We confirmed isolation of some minor tRNAs, such as tRNASec. Doublet bands of several tRNAs were found to be conformers originating from the same tRNA. Lower bands were degraded fragments. From 16 mg total RNA, total yields of isolated RNAs were ∼20–120 μg (Table 1). The binding capacities of the tip-columns limited the total yield of some abundant tRNAs. Half of the target RNAs showed over 80% purity, although purities of several RNAs were low due to inappropriate probe designs (Table 1) which were improved by redesigning probe sequences (data not shown)Figure 4.


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)

Automated parallel isolation of all species of E. coli tRNA and four sRNAs. (A) Polyacrylamide gel electrophoresis of E. coli tRNAs, 4.5S and 6S RNAs isolated by the RCC instrument. RNAs were visualized by ethidium bromide staining. Several tRNAs migrated as doublet bands, which were conformers of the same tRNAs. Species of tRNAs are shown as single letter abbreviations for the corresponding amino acid and a number extension in the case of multiple tRNAs for the same amino acid. E. coli total RNA (asterisk) was used as a marker. (B) Northern blot analysis of DsrA and SraH sRNA. Strong signals were observed in the eluted fraction (elution), weak signals were visible in the starting sample (load), and no signals were found in the flow through fraction (FT). Ethidium bromide (EtBr) staining is also shown. Most of the target RNAs were highly concentrated in the eluted fractions.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 4: Automated parallel isolation of all species of E. coli tRNA and four sRNAs. (A) Polyacrylamide gel electrophoresis of E. coli tRNAs, 4.5S and 6S RNAs isolated by the RCC instrument. RNAs were visualized by ethidium bromide staining. Several tRNAs migrated as doublet bands, which were conformers of the same tRNAs. Species of tRNAs are shown as single letter abbreviations for the corresponding amino acid and a number extension in the case of multiple tRNAs for the same amino acid. E. coli total RNA (asterisk) was used as a marker. (B) Northern blot analysis of DsrA and SraH sRNA. Strong signals were observed in the eluted fraction (elution), weak signals were visible in the starting sample (load), and no signals were found in the flow through fraction (FT). Ethidium bromide (EtBr) staining is also shown. Most of the target RNAs were highly concentrated in the eluted fractions.
Mentions: Using a 16 mg sample of total RNA in a common reservoir as the starting material, 6 runs were conducted in which 8 different tip-columns were attached to the head of the RCC instrument for purification of the ncRNAs. Pipetting with the RNA solution in the reciprocal circulation was repeated 40 times at 66°C. Then, columns were sequentially washed and eluted by pipetting three times in 12 sets of washing tubes and 6 sets of eluting tubes at 40 and 68°C, respectively. Sufficient washing was confirmed by measuring the optical densities at 260 nm of washed fractions (not over 0.01 A260 in last tubes). Isolated RNAs collected from the eluting tubes were electrophoresed in 10% denaturing polyacrylamide gels (Figure 4A). All tRNA species were visible on the gel as a single band or as major bands with some contamination. We confirmed isolation of some minor tRNAs, such as tRNASec. Doublet bands of several tRNAs were found to be conformers originating from the same tRNA. Lower bands were degraded fragments. From 16 mg total RNA, total yields of isolated RNAs were ∼20–120 μg (Table 1). The binding capacities of the tip-columns limited the total yield of some abundant tRNAs. Half of the target RNAs showed over 80% purity, although purities of several RNAs were low due to inappropriate probe designs (Table 1) which were improved by redesigning probe sequences (data not shown)Figure 4.

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