RNA analysis by ion-pair reversed-phase high performance liquid chromatography

Abstract

Ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) is presented as a new, superior method for the analysis of RNA. IP RP HPLC provides a fast and reliable alternative to classical methods of RNA analysis, including separation of different RNA species, quantification and purification. RNA is stable under the analysis conditions used; degradation of RNA during the analyses was not observed. The versatility of IP RP HPLC for RNA analysis is demonstrated. Components of an RNA ladder, ranging in size from 155 to 1770 nt, were resolved. RNA transcripts of up to 5219 nt were analyzed, their integrity determined and they were quantified and purified. Purification of mRNA from total RNA is described, separating mouse rRNA from poly(A)(+) mRNA. IP RP HPLC is also suitable for the separation and purification of DIG-labeled from unlabeled RNA. RNA purified by IP RP HPLC exhibits improved stability.

Figure 1

Separation of the components of an RNA ladder by IP RP HPLC. An RNA ladder (1 µg) containing fragments ranging in size from 155 to 1770 nt was analyzed by IP RP HPLC using gradient condition 1.

Figure 2

Effect of analysis temperature on resolution of RNA fragments. The RNA ladder shown in Figure 1 was analyzed by IP RP HPLC at three different temperatures using gradient condition 1. Analysis of the ladder at 75°C is shown in Figure 1.

Figure 3

Analysis of RNA transcript integrity by IP RP HPLC. (A) RNA transcripts ranging in size from 749 to 2971 nt were analyzed individually by IP RP HPLC using gradient condition 2. The superimposed chromatograms of four individual transcripts are shown. (B) A crude transcription reaction product is shown. The full-length transcription product has a length of 5219 nt. Analysis was performed under elution condition 2.

Figure 3

Analysis of RNA transcript integrity by IP RP HPLC. (A) RNA transcripts ranging in size from 749 to 2971 nt were analyzed individually by IP RP HPLC using gradient condition 2. The superimposed chromatograms of four individual transcripts are shown. (B) A crude transcription reaction product is shown. The full-length transcription product has a length of 5219 nt. Analysis was performed under elution condition 2.

Figure 4

RNA quantification after IP RP HPLC by peak integration. (A) Superimposed chromatograms of known amounts of MS2 RNA analyzed by IP RP HPLC under gradient condition 2. (B) Standard curve for MS2 RNA quantification, plot of integrated peak area from (A) versus injected amount of MS2 RNA (Table 1). The square of the Pearson product moment correlation coefficient (R²) through the given data points is 0.9985.

Figure 4

RNA quantification after IP RP HPLC by peak integration. (A) Superimposed chromatograms of known amounts of MS2 RNA analyzed by IP RP HPLC under gradient condition 2. (B) Standard curve for MS2 RNA quantification, plot of integrated peak area from (A) versus injected amount of MS2 RNA (Table 1). The square of the Pearson product moment correlation coefficient (R²) through the given data points is 0.9985.

Figure 5

Separation and purification of unlabeled and labeled β-actin RNA transcripts. DIG-labeled β-actin RNA transcript (1 µg) was analyzed by IP RP HPLC under elution condition 3. Unlabeled β-actin RNA elutes with a retention time of ∼4.7 min. DIG-labeled β-actin RNA elutes at the end of the gradient. Each peak was captured by manual collection.

Figure 6

Mouse brain total RNA analyzed by IP RP HPLC. All RNA samples were analyzed by IP RP HPLC using gradient condition 1. (A) Mouse brain total RNA (20 µg). (B) Analysis of large rRNAs (a mixture of co-eluting 28S and 18S rRNA, respectively) purified by peak capture from total RNA. (C) Analysis of mRNA purified by peak capture from total RNA. (D) Analysis of mouse brain poly(A)⁺ mRNA (5 µg) obtained after two rounds of oligo(dT)–cellulose purification.

Figure 6

Mouse brain total RNA analyzed by IP RP HPLC. All RNA samples were analyzed by IP RP HPLC using gradient condition 1. (A) Mouse brain total RNA (20 µg). (B) Analysis of large rRNAs (a mixture of co-eluting 28S and 18S rRNA, respectively) purified by peak capture from total RNA. (C) Analysis of mRNA purified by peak capture from total RNA. (D) Analysis of mouse brain poly(A)⁺ mRNA (5 µg) obtained after two rounds of oligo(dT)–cellulose purification.

Figure 6

Mouse brain total RNA analyzed by IP RP HPLC. All RNA samples were analyzed by IP RP HPLC using gradient condition 1. (A) Mouse brain total RNA (20 µg). (B) Analysis of large rRNAs (a mixture of co-eluting 28S and 18S rRNA, respectively) purified by peak capture from total RNA. (C) Analysis of mRNA purified by peak capture from total RNA. (D) Analysis of mouse brain poly(A)⁺ mRNA (5 µg) obtained after two rounds of oligo(dT)–cellulose purification.

Figure 6

Mouse brain total RNA analyzed by IP RP HPLC. All RNA samples were analyzed by IP RP HPLC using gradient condition 1. (A) Mouse brain total RNA (20 µg). (B) Analysis of large rRNAs (a mixture of co-eluting 28S and 18S rRNA, respectively) purified by peak capture from total RNA. (C) Analysis of mRNA purified by peak capture from total RNA. (D) Analysis of mouse brain poly(A)⁺ mRNA (5 µg) obtained after two rounds of oligo(dT)–cellulose purification.