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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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Automated parallel isolation of several ncRNAs from yeast and mouse. (A) Polyacrylamide gel electrophoresis of U4 and U6 snRNAs, SCR1 (SRP RNA), 4 snoRNAs (SNR5, SNR9, SNR128 and SNR190) and mitochondrial tRNAMet, which were isolated by RCC. The gel is stained with SYBR Green II (Invitrogen). Target RNAs are indicated by arrow heads. (B) Isolated mouse ncRNAs: 7SK RNA, SRP RNA, RNaseP RNA, RNaseMRP RNA, Y1 RNA, Vault RNA and U5 snRNA. Target RNAs are indicated by arrow heads.
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Figure 5: Automated parallel isolation of several ncRNAs from yeast and mouse. (A) Polyacrylamide gel electrophoresis of U4 and U6 snRNAs, SCR1 (SRP RNA), 4 snoRNAs (SNR5, SNR9, SNR128 and SNR190) and mitochondrial tRNAMet, which were isolated by RCC. The gel is stained with SYBR Green II (Invitrogen). Target RNAs are indicated by arrow heads. (B) Isolated mouse ncRNAs: 7SK RNA, SRP RNA, RNaseP RNA, RNaseMRP RNA, Y1 RNA, Vault RNA and U5 snRNA. Target RNAs are indicated by arrow heads.

Mentions: Eukaryotic ncRNAs were chosen for the next validation of the RCC method. In Saccharomyces cerevisiae, 8 probes were designed for U4 and U6 snRNAs, SRP RNA (SCR1), 4 species of snoRNAs and mitochondrial tRNAMet (Table 2). As shown in Figure 5, all target RNAs, including the long RNA SCR1 (522 nt), were successfully isolated by this method. From 40 mg total RNA, the yields of isolated RNAs were ∼0.16–3.24 μg (Table 2). Seven mouse ncRNAs targets were selected: 7SK, SRP, RNase P, RNase MRP, Y1, Vault and U5 (Table 2). As shown in Figure 5B, each target RNA was isolated by RCC and the yields of isolated RNAs were ∼0.08–2.56 μg, from a starting sample of 4 mg total RNA (Table 2).Table 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)

Automated parallel isolation of several ncRNAs from yeast and mouse. (A) Polyacrylamide gel electrophoresis of U4 and U6 snRNAs, SCR1 (SRP RNA), 4 snoRNAs (SNR5, SNR9, SNR128 and SNR190) and mitochondrial tRNAMet, which were isolated by RCC. The gel is stained with SYBR Green II (Invitrogen). Target RNAs are indicated by arrow heads. (B) Isolated mouse ncRNAs: 7SK RNA, SRP RNA, RNaseP RNA, RNaseMRP RNA, Y1 RNA, Vault RNA and U5 snRNA. Target RNAs are indicated by arrow heads.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 5: Automated parallel isolation of several ncRNAs from yeast and mouse. (A) Polyacrylamide gel electrophoresis of U4 and U6 snRNAs, SCR1 (SRP RNA), 4 snoRNAs (SNR5, SNR9, SNR128 and SNR190) and mitochondrial tRNAMet, which were isolated by RCC. The gel is stained with SYBR Green II (Invitrogen). Target RNAs are indicated by arrow heads. (B) Isolated mouse ncRNAs: 7SK RNA, SRP RNA, RNaseP RNA, RNaseMRP RNA, Y1 RNA, Vault RNA and U5 snRNA. Target RNAs are indicated by arrow heads.
Mentions: Eukaryotic ncRNAs were chosen for the next validation of the RCC method. In Saccharomyces cerevisiae, 8 probes were designed for U4 and U6 snRNAs, SRP RNA (SCR1), 4 species of snoRNAs and mitochondrial tRNAMet (Table 2). As shown in Figure 5, all target RNAs, including the long RNA SCR1 (522 nt), were successfully isolated by this method. From 40 mg total RNA, the yields of isolated RNAs were ∼0.16–3.24 μg (Table 2). Seven mouse ncRNAs targets were selected: 7SK, SRP, RNase P, RNase MRP, Y1, Vault and U5 (Table 2). As shown in Figure 5B, each target RNA was isolated by RCC and the yields of isolated RNAs were ∼0.08–2.56 μg, from a starting sample of 4 mg total RNA (Table 2).Table 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