© 2001 Oxford University Press

Nucleic Acids Research, 2001, Vol. 29, No. 2 e7

RNA analysis by ion-pair reversed-phase highperformance liquid chromatography
Arezou Azarani* and Karl H. Hecker
Transgenomic Inc., 2032 Concourse Drive, San Jose, CA 95131, USA
Received August 16, 2000; Revised November 1, 2000; Accepted November 10, 2000

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 classicalmethods of RNA analysis, including separation ofdifferent RNA species, quantification and purification.
RNA is stable under the analysis conditions used;degradation of RNA during the analyses was notobserved. 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, theirintegrity determined and they were quantified andpurified. Purification of mRNA from total RNA isdescribed, separating mouse rRNA from poly(A)+mRNA. IP RP HPLC is also suitable for the separationand purification of DIG-labeled from unlabeled RNA.
RNA purified by IP RP HPLC exhibits improved
stability.

The analysis of DNA by ion-pair reversed-phase highperformance liquid chromatography (IP RP HPLC) under nondenaturing, partially denaturing or fully denaturing conditions
has gained widespread acceptance over the past years. IP RP
HPLC under non-denaturing conditions provides a means forsequence-independent sizing of DNA fragments of up to 2000 bp(7,8). The main impact of HPLC performed under partiallydenaturing conditions has been in mutation detection by meansof heteroduplex analysis (9,10). IP RP HPLC under fully
denaturing conditions is described in the literature for analysisof oligonucleotides (11,12) and single-stranded (ss)DNA fragmentsof up to 100 nt (13). Under fully denaturing conditions separationof ssDNA on an alkylated poly(styrene divinylbenzene) matrix
is sequence dependent. Separation characteristics of RNA are
expected to resemble those of ssDNA under fully denaturingconditions.
In this report the use of IP RP HPLC technology under fully
denaturing conditions is extended to RNA analysis. This
technology can be applied to qualification, quantification andpurification of a wide range of labeled/unlabeled RNA
samples, such as discrete transcripts, rRNA, mRNA and total
RNA. The technology is shown to offer a significant improvement over current methods of RNA analysis.

INTRODUCTION
A number of labor intensive and time consuming techniques
are currently available for RNA isolation, purification andquantification. Quantification of RNA samples is performedby measuring their absorption at 260 nm, while the quality andintegrity of RNA samples are generally determined by gelelectrophoresis followed by ethidium bromide visualization(1–3). Absorption measurements, however, do not provide
information about the integrity (impurities and degradation) of
the RNA sample analyzed. Analysis of RNA by electrophoretic techniques creates the potential for RNA degradation
by exogenous RNases due to prolonged handling. Neither oneof the above methods is suitable for RNA purification. Furthermore, aliquots of RNA analyzed by either procedure are notrecoverable for use in downstream applications.
Currently the only mRNA purification technology available
is that of oligo(dT) selection (1,2). A number of hybridizationand affinity-based methods are available for the isolation andpurification of labeled or unlabeled specific mRNA species (4–6). However, recoveries for purified mRNA species obtainedwith these procedures are generally low and the mRNA recovered often exhibits varying degrees of purity and integrity (dueto the presence of degraded RNA, proteins or genomic DNA).

MATERIALS AND METHODS
Materials
The WAVE® System with DNASep® cartridges (7.8 × 50 mm)
and triethylammonium acetate (TEAA) was provided byTransgenomic Inc. (San Jose, CA). RNA ladders werepurchased from Gibco BRL (Rockville, MD). Total RNA andmRNA samples were obtained from Clontech (Palo Alto, CA).
In vitro transcription kits for RNA synthesis were purchasedfrom Ambion (Austin, TX). Digoxigenin (DIG)-labeled β-actin
RNA, the Titan reverse transcription PCR kit and MS2
bacteriophage RNA were purchased from Roche Molecular
Biochemicals (Indianapolis, IN). The β-actin-specific reverse
transcription PCR primers used were 5′-GTC GAC AAC GGC
TCC GGC ATG-3′ and 5′-GGA TCT TCA TGA GGT AGT
CAG-3′, yielding a 550 bp product.
HPLC analysis
All RNA samples were analyzed by IP RP HPLC on theWAVE System using a DNASep cartridge. The stationary
phase of the cartridge consists of a non-porous, alkylatedpoly(styrene divinylbenzene) matrix. Chromatography was*To whom correspondence should be addressed. Tel: +1 408 432 3230; Fax: +1 408 432 8910; Email: aazarani@transgenomic.com

e7 Nucleic Acids Research, 2001, Vol. 29, No. 2

PAGE 2 OF 9

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.

performed using a two eluent buffer system. Buffer A consistsof an aqueous solution of 0.1 M TEAA, pH 7.0, and buffer B
consists of an aqueous solution of 0.1 M TEAA, pH 7.0, with25% (v/v) acetonitrile. RNA analyses were performed underfully denaturing conditions at 75°C. Sizing of dsDNA was
performed under non-denaturing conditions at 50°C. Chromatograms were recorded at a wavelength of 260 nm. Quantificationof peaks was performed by integration of peak areas. RNA for
downstream uses was purified by peak capture. IP RP HPLC
analyses were performed using the following gradient conditions:condition 1, flow rate 0.9 ml/min, 38–40% B over 1.0 min, to
60% B over 15 min, to 66% B over 6.0 min, to 70% B over
0.5 min, to 100% B over 0.5 min, held at 100% B for 1 min, to
38% B over 1 min, held at 38% B for 2 min; condition 2, flowrate 0.9 ml/min, 38–60% B over 30 min, to 100% B over 2 min,to 38% B over 3 min; condition 3, flow rate 0.9 ml/min, 38–40%
B over 1 min, to 60% B over 3 min, to 100% B over 1 min, held
at 100% B for 6 min, to 38% B over 1 min, held at 38% B for
1 min.
RNA purification
RNA was purified by manual peak collection. Fractions werecollected for 1 min, corresponding to a fraction volume of
0.9 ml at a flow rate of 0.9 ml/min. Collected RNA samples
may be precipitated or used directly in the collection buffer fordownstream uses. For reverse transcription PCR studies 5 µl
aliquots of collected fractions were used directly. Precipitationof eluted RNA samples was by addition of 10% (v/v) precipitation buffer (10 mM Tris–HCl, pH 7.0, 1 mM EDTA, 3.0 M
NaCl), 1% (v/v) glycogen (10 mg/ml) and 2.5 vol ethanol.
Samples were kept at –70°C for 10 min or at –20°C for 2 h
before centrifugation at 13 000 r.p.m. for 15 min at 4°C. All
precipitated RNA fractions were reconstituted in DEPCtreated water.

RESULTS AND DISCUSSION
Resolution of discrete RNA fragments and determinationof RNA integrity
IP RP HPLC under fully denaturing conditions with on-line
UV detection offers a sensitive and reliable method for thedetection and analysis of RNA transcripts and size markers.
The integrity of RNA is not compromised under the analysis
conditions used, 75°C and elution buffers containing TEAA
and acetonitrile. Figure 1 shows the chromatogram of an RNA
ladder containing fragments ranging in size from 155 to 1770 nt.
The integrity of the individual fragments is apparent from theirwell-defined peak shape. Degradation of RNA, which would
result in the appearance of spurious peaks in the chromatogram, is
not observed.
Recently, Georgopoulos and Leibowitz (14) reported thefractionation of ssRNA molecules ranging in size from 200 to1000 nt by IP RP HPLC. The authors claim that in the size
range 400–1000 nt retention times are strictly size dependent.
However, they do note that chromatographic mobility ofssRNA molecules may be determined by other factors in
addition to size. This concession is prompted by the observationof aberrant retention times of some of the RNA molecules.
Oefner reported a clear sequence dependence of retention time
for ssDNA molecules (<100 nt in length) analyzed by IP RP
HPLC under fully denaturing conditions (7). Based on thereports by Georgopoulos and Leibowitz (14) as well as Oefner(7), retention times for ssRNA fragments were expected to besequence dependent. We analyzed various RNA size markers
and transcripts of known sequence and were able to confirm
the sequence dependence of retention time for RNA fragments(data not shown).
Oefner (13) recently reported a strong effect of analysis temperature on resolution for ssDNA fragments. Resolution generally

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Nucleic Acids Research, 2001, Vol. 29, No. 2 e7

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.

improved with increasing temperature. Thus, the effect oftemperature on resolution was attributed to the disruption of
intra- and intermolecular interactions. In agreement with
Oefner’s observations for ssDNA, we noticed a decrease in
resolution for RNA at lower analysis temperatures (Fig. 2).
Consequently, all RNA analyses were performed at 75°C.
The ability to separate RNA fragments by IP RP HPLC
under fully denaturing conditions can be used to determine theintegrity and purity of RNA transcripts. In order to demonstrate this, different plasmid vectors were digested withrestriction endonucleases and transcribed to generate specific
RNA transcripts of known length and sequence. Each individualtranscript was analyzed by IP RP HPLC under fully denaturingconditions to determine its quality and purity. Several superimposed chromatograms of individual transcripts are shown in
Figure 3A. The purity and integrity of each transcript inFigure 3A is apparent from the presence of a single peak.
RNA undergoing degradation is broken down into smaller
fragments. These breakdown products are retained lessstrongly on the cartridge and result in peaks that elute at shorter
retention times. Early termination products of transcription
also result in shorter RNA fragments, which likewise eluteprior to the full-length RNA transcript. The detection of RNA
degradation and spurious transcription products is shown in
Figure 3B, which shows the analysis of a crude 5219 nt RNAtranscript. Peaks preceding the full-length 5219 nt transcript

indicate the presence of spurious transcription and/or degradation
products in this crude transcription reaction. Therefore, IP RP
HPLC may be used to assess the quality and integrity of transcripts. Furthermore, chromatography has the advantage overother technologies that samples may be recovered by peakcapture. Thus, IP RP HPLC can also be used for transcript
purification.
RNA quantification
IP RP HPLC provides a reliable and accurate method for RNA
quantification by peak integration. RNA quantities per peak in
the low nanogram range can be detected. The largest amount ofRNA loaded for an individual peak in Figure 4A is 1000 ng.
Amounts of total RNA of up to 100 µg have been analyzed andfractionated on the cartridge used here (see below). Analysis
and quantification of MS2 RNA, which is 3569 nt long, isshown in Figure 4. Amounts loaded ranged from 8 to 1000 ng.
RNA quantification was performed by peak integration.
Results are shown in Table 1 and graphed in Figure 4B. A
correlation factor of 1250 mVs/ng RNA was used for the determination of RNA quantities. This factor is dependent on flowrate and may also vary between instruments. The procedure fordetermining the correlation factor is described in Transgenomic Application Note 115 (http://www.transgenomic.com/
pdf/AN115.pdf ) for DNA. Figure 4 shows that peak area of ane7 Nucleic Acids Research, 2001, Vol. 29, No. 2

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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-lengthtranscription product has a length of 5219 nt. Analysis was performed under elution condition 2.

individual fragment increases linearly with the amount of RNA
analyzed from the low nanogram range to at least 1000 ng.
Purification of unlabeled and DIG-labeled RNA transcripts
RNA fragments separated by IP RP HPLC can be recovered after
analysis. Figure 5 shows the separation of DIG-labeled β-actin

RNA standard (588 nt) from unlabeled β-actin RNA transcript,
which was present in small amounts. Two well-resolved peaks
corresponding to unlabeled (retention time 4.7 min) and DIGlabeled (retention time 7.8 min) β-actin RNA are observed.
Retention times of nucleic acids on the DNASep cartridge aredetermined by the hydrophobicity of the molecules analyzed

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Nucleic Acids Research, 2001, Vol. 29, No. 2 e7

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.

(9,10). The β-actin RNA labeled with the hydrophobic DIG tag
shows a greater retention time than unlabeled β-actin RNA.
Individual peaks were captured by manual collection. The

presence and integrity of captured β-actin RNA, both unlabeled and labeled, was confirmed by reverse transcription–PCRusing 5 µl aliquots of each collected sample. A 550 bp PCR

e7 Nucleic Acids Research, 2001, Vol. 29, No. 2

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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.

product was successfully amplified from the labeled and unlabeled β-actin RNA fractions. Fractions collected before 4 orafter 9 min during the run served as negative controls and did
not produce a 550 bp PCR product (data not shown).

Table 1. MS2 RNA quantification results
MS2 RNA injected(ng)

Integrated peakarea (mVs)

Calculated RNAamount (ng)

8

8062

6.4

16

20 38016.3

25

28 24522.6

125

140 173

112.1

250

272 300

217.8

500

628 035

502.4

1000

1 292 338

1033.8RNA samples collected during IP RP HPLC were morestable in the collection buffer than expected. RNA purified bypeak capture was stable for days when kept at room temperatureand for months when kept at –20 or –70°C. Stability of RNA
after collection was assessed by gene-specific reverse transcription–PCR on mRNA template after purification by IP RP
HPLC (manuscript in preparation). Our explanation for thisobservation is that since proteins have a very low affinity forthe alkylated poly(styrene divinylbenzene) matrix under theelution conditions used for RNA analysis, they are hardly
retained during IP RP HPLC. RNases and other proteins eluteat an early stage in the gradient and are removed from RNA
samples during analysis. Thus, RNA purified by IP RP HPLC
exhibits improved stability.
Purification of mRNA from total RNA by IP RP HPLCIP RP HPLC can be used for the separation and purification ofrRNA and mRNA. An analysis of mouse brain total RNA(Clontech, Palo Alto, CA) on a DNASep cartridge is shown in
Figure 6A. The 5S, 18S (1900 nt) and 28S (4700 nt) rRNA
fragments elute before the bulk of mRNA fragments. It waspointed out above that retention times of RNA fragments aresequence dependent. However, the reasons for this apparent
separation of rRNA and mRNA require some explanation. The
alkylated, hydrophobic column matrix interacts directly withthe hydrophobic alkyl chains of the TEAA contained in theelution buffer. The positively charged ammonium ions in turninteract with the negatively charged phosphate backbone of
nucleic acids, thereby mediating the interaction between
nucleic acids and column matrix. In addition to the latter interactions, the exposed bases of single-stranded nucleic acids
themselves interact with the column matrix due to their hydrophobic character. Different bases exhibit different degrees ofhydrophobicity. This is the basis for the sequence dependenceof retention time in IP RP HPLC under the analysis conditions
described here. Hydrophobicity increases in the order C < G <T < A (7). Thus, it is expected that single-stranded nucleic acidmolecules with a high adenine content exhibit longer retention
times than those with high guanine or cytosine content. Polyadenylated mRNA is therefore retained more strongly on the
DNASep cartridge than rRNA, which is not polyadenylated.
Figure 6B and C shows rRNA and mRNA purified by peakcapture. Fractions containing rRNA and mRNA werecollected. RNA was precipitated, resuspended in 20 µl of

PAGE 7 OF 9

DEPC-treated water and re-analyzed by IP RP HPLC. Using
primers specific for 18S rRNA (Ambion, Austin, TX) a 495 bp
product was successfully amplified from rRNA fractions.
Using primers specific for β-actin message a 550 bp PCR
product was amplified from mRNA fractions (data not shown).
rRNA purified by IP RP HPLC contains only traces of mRNAand purified mRNA contains only trace amounts of rRNA. We
concede that some mRNA species, mainly shorter mRNA
species, are lost in this purification procedure due to co-elution

Nucleic Acids Research, 2001, Vol. 29, No. 2 e7

with rRNA. However, we point out that the bulk of the mRNA,
particularly longer mRNA species, is captured.
The hydrophobicity ratios calculated for both 18S and 28S
rRNA were 0.8 and 0.5, respectively. From these ratios it was
expected that 28S should elute before 18S rRNA in Figure 6A.
Performing reverse transcription–PCR studies on the collectedrRNA fractions (using gene-specific primers to 18S and 28S
RNA) it was concluded that this is indeed the case. However,these studies also indicated that under the gradient conditions

e7 Nucleic Acids Research, 2001, Vol. 29, No. 2

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Figure 6. (Previous page and above) 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.

used here the two rRNA peaks are not well resolved, i.e. thepeaks for the 28S and 18S rRNA show significant overlap.
Thus, the rRNA peaks in Figure 6A and B are labeled as largerRNAs.
For comparative purposes mouse brain mRNA (Clontech),which was isolated by two rounds of poly(A)+ RNA selection,
was analyzed by IP RP HPLC. As shown in Figure 6D, thissample still contained some rRNA contamination (confirmedby reverse transcription–PCR analysis) after two rounds ofpoly(A)+ selection. Significantly less rRNA contamination isobserved after only a single round of purification by IP RP
HPLC (Fig. 7C). Recovery of RNA after IP RP HPLC is generally
higher than that after oligo(dT)–cellulose purification. Purificationof RNA samples by IP RP HPLC can be performed in a matter

PAGE 9 OF 9of minutes. Classical methods of mRNA purification require
several hours of labor intensive manipulation.
In conclusion, the new technology presented here for RNA
analysis and purification combines the high resolution
capability of IP RP HPLC, the high sensitivity of on-line UV
detection and ease of sample recovery to provide a reliable andfast alternative to current, conventional methods of RNA analysis.
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