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`METHOD
`
`Enrichment and analysis of RNA centered on ion
`pair reverse phase methodology
`
`MARK J. DICKMAN1 and DAVID P. HORNBY2
`1Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield,
`Sheffield S1 3JD, United Kingdom
`2Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom
`
`ABSTRACT
`
`Here we describe a procedure for the rapid enrichment of RNA from cell extracts and the subsequent fractionation and analysis
`of the ‘‘small RNA’’ population by ion pair reverse phase chromatography. Solid phase extraction procedures have been
`developed utilizing nonporous alkylated poly(styrene- divinylbenzene) particles in conjunction with ion pair reagents to enrich
`total RNA. This approach facilitates the selective enrichment and separation of the relatively lower abundance small RNAs, from
`the more abundant higher molecular weight rRNA species. We also describe the application of monolithic capillaries in
`conjunction with ion pair reverse phase chromatography to bring increased sensitivity in the analysis of very low abundance
`RNAs. These approaches will simplify the biochemical analysis of this class of molecules, which are emerging as important
`regulators of global gene expression in higher organisms.
`
`Keywords: ion pair reverse phase chromatography; solid phase extraction; total RNA; small RNAs and miRNA
`
`INTRODUCTION
`
`Our appreciation of the relative importance of RNA in
`numerous biological processes has increased substantially
`over recent years. The discoveries of catalytic RNA (Kruger
`et al. 1982; Guerrier-Takada et al. 1983), RNA interference
`and noncoding regulatory RNAs (for review, see Ambros
`2004; Bartel 2004) have impacted a broad range of disci-
`plines. The development of methodology for the isolation
`and characterization of biological RNA has had a somewhat
`checkered history alongside comparable methods for the
`analysis of
`its macromolecular counterparts, DNA and
`proteins. Moreover, the extraction, isolation, and analysis of
`RNA is routinely more difficult in comparison to that
`required for DNA. In approaching the problem of RNA
`isolation, the stability and molecular heterogeneity are of
`immediate concern. RNA is susceptible to endo- and exo-
`nuclease mediated degradation, rendering the initial stages
`of extraction and the downstream storage of the purified
`material more challenging than for DNA. Furthermore,
`
`Reprint requests to: Mark J. Dickman, Biological and Environmental
`Systems Group, Department of Chemical and Process Engineering, Univer-
`sity of Sheffield, Sheffield S1 3JD, United Kingdom; e-mail: m.dickman@
`sheffield.ac.uk; fax: +0114-222-7566.
`Article published online ahead of print. Article and publication date are
`at http://www.rnajournal.org/cgi/doi/10.1261/rna.2278606.
`
`downstream analysis of RNA requires separations to be
`typically performed under harsh denaturing conditions.
`Current total RNA isolation procedures use a combi-
`nation of denaturing agents, acid phenol chloroform ex-
`traction followed by precipitation of
`the nucleic acids
`(Chomczynski and Sacchi 1987). This procedure is time-con-
`suming and is particularly inefficient with respect to the
`recovery of ‘‘small’’ RNAs. In addition, the use of glass filters
`that bind RNA in the presence of chaotropic salts have been
`used to isolate and purify total RNA from cell extracts and
`tissues (Boom et al. 1990). Proteins and DNA are removed by
`washing the filter, and RNA is subsequently eluted. Low RNA
`yields are often obtained due to overloading of the column,
`which can cause the column to clog or can prevent the
`RNA from binding efficiently. Moreover, the recovery of
`small RNAs using traditional approaches is inefficient and
`the fractionation of RNA species is not always possible.
`Following enrichment of an RNA species, downstream
`analysis is necessary to determine such properties as molec-
`ular weight and primary, secondary, and tertiary structure
`of the RNA. A variety of approaches have been developed
`centered around gel electrophoresis under denaturing con-
`ditions, with the aim of disrupting RNA secondary struc-
`ture to provide accurate molecular weight determinations
`(Sambrook et al. 1989). Many of the current methodologies
`utilizing denaturing conditions incorporate the use of toxic,
`hazardous chemicals such as glyoxal/dimethyl sulfoxide and
`
`RNA (2006), 12:691–696. Published by Cold Spring Harbor Laboratory Press. Copyright ª 2006 RNA Society.
`
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`Dickman and Hornby
`
`methyl mercury hydroxide (Bailey and Davidson 1976; Mc-
`Master and Carmichael 1977; Sebo and Schmit 1982). Alterna-
`tive denaturants include urea, formamide, and formaldehyde
`(Floyd et al. 1974; Pinder et al. 1974; Lehrach et al. 1977). There
`are additional problems associated with the use of such re-
`agents, including difficulty in staining the gels and interference
`with the electrophoretic mobility of the RNA species (Tsang
`et al. 1993).
`
`Ion pair reverse phase chromatography
`
`Ion pair reverse phase high-performance liquid chromato-
`graphy (IP RP HPLC) on nonporous alkylated poly(sty-
`rene-divinylbenzene) particles combines very high-resolution
`separation of nucleic acids with very short analysis times,
`therefore offering significant advantages and opportunities
`for the analysis of nucleic acids (Gjerde et al. 2002) over
`electrophoresis methods. The nonporous polymeric media,
`in conjunction with the highly monodisperse nature of the
`particles, results in the minimization of the diffusion paths
`(Huber et al. 1993; Huber 1998). Furthermore, the media is
`robust, resistant to a broad range of temperatures and pH.
`When a sample is injected, the anions from the nucleic acid
`combine with the oppositely charged ammonium cations of
`the ion pair reagent. The most common ion pair reagent is
`triethylammonium acetate (TEAA). The alkyl groups of such
`ion pair reagents enable the nucleic acids to become hydro-
`phobic and the molecule adsorbs to the stationary phase. The
`longer the alkyl chain used, the more hydrophobic the nucleic
`acid and the stronger the interaction with the stationary phase.
`In addition, larger nucleic acids have a stronger interaction
`with the stationary phase as the increased length enables an
`increased number of ion pair molecules to be associated with
`the nucleic acid. During the HPLC a gradient of acetonitrile is
`started. As the acetonitrile concentration is increased this
`causes the smaller nucleic acids to desorb from the stationary
`phase first. Finally as the acetonitrile concentration is further
`increased the larger nucleic acids are desorbed and travel
`down the column to the detector.
`Given the complexity of the RNA population from even
`the simplest of organisms, it is critical that measures be
`taken to preserve that population prior to separation (or
`indeed any form of analysis). Separation and analysis using
`ion pair reverse phase chromatography is performed using a
`combination of ion pairing reagent (TEAA at pH 7.4) and
`acetonitrile at elevated temperatures. Under such condi-
`tions RNA maintains its chemical integrity and biological
`activity (Azarani and Hecker 2001). Furthermore, when the
`effect of acetonitrile upon the activity of a typical ribo-
`nuclease enzyme was investigated at a range of solvent
`concentrations and temperatures, a marked effect upon
`
`C in
`the stability of the enzyme was observed. Thus at 60
`20% acetonitrile, Bovine RNase is reversibly unfolded,
`while at 60% acetonitrile at the same temperature the en-
`zyme is unfolded but in a reversible manner (M.J. Conroy,
`
`692
`
`RNA, Vol. 12, No. 4
`
`D.P. Hornby, and M.J. Dickman, unpubl.). In addition, the
`mechanism of separation using IP RP HPLC also serves to
`eliminate contaminating RNases during the chromatogra-
`phy. Under the conditions used, the RNA is retained on
`the stationary phase allowing separation from the RNases.
`These observations suggest that the elimination of the dele-
`terious effects of contaminating RNases enables the puri-
`fication and analysis of high-quality RNA using IP RP
`methodology.
`Here we describe the enrichment of RNA, including the
`subsequent fractionation of small RNAs in conjunction
`with the rapid analysis of the isolated purified RNA. The
`methodology and analysis is centered on ion pair reverse
`phase chromatography. Solid phase extraction procedures
`have been developed utilizing nonporous alkylated poly-
`(styrene-divinylbenzene) particles in conjunction with ion
`pair reagents to enrich total RNA. This methodology
`enables the selective enrichment of the lower abundance
`small RNAs from the larger, more abundant rRNA species.
`In addition, rapid analysis of the RNA fractions isolated
`using the solid phase extraction procedures using ion pair
`reverse phase chromatography has been performed. These
`results demonstrate the successful enrichment of small
`RNAs and in addition facilitate the analysis of small RNAs
`and larger rRNA species during a single chromatographic
`analysis. RNA analysis has also been extended using mono-
`lithic capillaries in conjunction with ion pair reverse phase
`chromatography, demonstrating higher sensitivity, and is
`ideal for the analysis of low concentrations of RNA samples
`and the subsequent downstream fractionation of the RNA
`species. Using such approaches allows the analysis of low
`nanogram quantities of RNA and furthermore enables the
`subsequent fraction of RNA in small volumes.
`
`RESULTS AND DISCUSSION
`
`Ion pair reverse phase chromatography of RNA
`under denaturing conditions
`
`When a total RNA preparation extracted from both pro-
`karyotic and eukaryotic cells is subjected to IP RP HPLC the
`resulting chromatogram is typified by that shown in Figure
`1. There are several distinct features within the profile; the
`earliest eluting species shown in the chromatogram are the
`tRNA (including possible mature miRNA), followed by
`the elution of the rRNA. Verification of the RNA species
`was performed by comparison with molecular weight stan-
`dards, purified tRNA and rRNA standards and using specific
`primers for RT PCR (data not shown). Standard gel electro-
`phoretic analysis of total RNA samples often requires the
`combined use of denaturing agarose and denaturing poly-
`acrylamide gels. The separation of the larger rRNA species
`is performed using denaturing agarose gel electrophore-
`sis, while the separation and analysis of the small RNAs
`requires denaturing polyacrylamide gels. These results dem-
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`RNA analysis centered on IP RP HPLC
`
`RNA species) and passed down a second solid phase
`extraction column. Under these ion pair conditions the
`small RNAs are retained by the stationary phase. In both
`cases the extraction columns are washed in the presence of
`the appropriate ion pair reagent and acetonitrile to
`remove salts and contaminants. The RNA species are sub-
`sequently eluted from the column by the addition of 50%
`acetonitrile. The results of the solid phase extraction pro-
`cedures are shown in Figure 2. Figure 2A shows the RNA
`species retained on the initial solid phase extraction col-
`umn. The results demonstrate that the rRNA species are
`retained while the small RNA species pass directly through
`the column. Figure 2B shows the eluted fraction following
`addition of the stronger ion pair reagent (tetrabutylam-
`monium bromide) to the unbound species. The results
`show that the small RNA species are now retained on the
`stationary phase and can be subsequently eluted with
`acetonitrile. Furthermore, the enriched small RNAs are
`completely free of any contaminating rRNA (see Fig.
`2B). These results demonstrate that alkylated poly(styr-
`ene-divinylbenzene) particles can be used successfully for
`the fractionation and purification of small RNAs from
`
`FIGURE 1. IP RP HPLC chromatogram of eukaryotic total RNA. The
`chromatogram shows the analysis of (3 mg) total RNA extracted from
`Hela cells using gradient 2 (see Materials and Methods). A shows the
`typical profile of the early eluting small RNA species and the later
`coeluting 18S and 23S rRNA. B shows an enhanced view of the smaller
`RNA species. The tRNA, 5.8S, and 5S rRNA are highlighted.
`
`onstrate that ion pair reverse phase chromatography en-
`ables the rapid analysis of total RNA, including the rapid
`separation and fractionation of tRNA, small rRNA, and large
`rRNA species during a single chromatographic analysis.
`
`Solid phase extraction and enrichment of small RNAs
`
`We have previously shown that using ion pair reverse
`phase chromatography in conjunction with alkylated
`poly(styrene-divinylbenzene) particles enables high-reso-
`lution separations of RNA and the rapid analysis of total
`RNA populations. We have used the same ion pair meth-
`odology to extract total RNA, and in particular, to specif-
`ically enrich the small RNAs from total RNA. Solid phase
`extraction columns were prepared using 8 mm alkylated
`poly(styrene-divinylbenzene) particles;
`the total RNA
`extracted from HeLa cells was adsorbed to the stationary
`phase in the presence of 0.2 M TEAA in 10% acetonitrile
`(pH 7). Under these conditions only the 18S and 23S rRNAs
`are adsorbed: The smaller RNA species are not retained. A
`stronger ion pair reagent (tetrabutylammonium bromide)
`is subsequently added to the unbound fraction (small
`
`FIGURE 2. Solid phase enrichment and fractionation of RNA using
`alkylated poly(styrene-divinylbenzene) particles. A shows the IP RP
`HPLC chromatogram of the enriched and fractionated large RNA
`species from total RNA extracts. B shows the IP RP HPLC chromato-
`gram of the enriched small RNA species in the absence of larger rRNA
`species.
`
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`Dickman and Hornby
`
`total RNA preparations, using ion pair methodology
`under stabilizing conditions.
`
`RNA analysis using capillary HPLC
`
`Capillary chromatography using ion pair reverse phase con-
`ditions on poly(styrene-divinylbenzene) monoliths has pre-
`viously been used for the separation of DNA, at very high
`resolution and over short analysis times with the advantage of
`increased sensitivity compared to traditional separations using
`conventional analytical columns (Oberacher and Huber 2002;
`Walcher et al. 2002). We have used 200 mm i.d. poly(styrene-
`divinylbenzene) monoliths in the analysis of RNA under
`denaturing conditions. The analysis of RNA using capillary
`chromatography was also extended to the analysis of total
`RNA extracted from HeLa cells: The results are shown in
`Figure 3. A similar elution profile of the RNA species is
`obtained as previously, with the early eluting small RNA
`followed by the coeluting rRNA species. The analysis using
`the 200 mm i.d. monoliths further illustrates the increase in
`sensitivity that can be achieved in the analysis of RNA. Nine
`nanograms (9 ng) of total RNA was analyzed in comparison
`
`FIGURE 3. IP RP mHPLC chromatogram of eukaryotic total RNA.
`The chromatogram shows the analysis of 9.2 ng total RNA extracted
`from Hela cells using gradient 4 (see Materials and Methods). A shows
`the typical profile of the early eluting small RNA species and the later
`coeluting 18S and 23S rRNA. B shows an enhanced view of the smaller
`RNA species. The tRNA, 5.8S, and 5S rRNA are highlighted.
`
`694
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`RNA, Vol. 12, No. 4
`
`to the microgram quantities of RNA required using conven-
`tional analytical columns to visualize the lower abundance
`small RNA and rRNAs. Analysis of the abundant rRNA spe-
`cies alone requires sub-nanogram quantities. In addition, frac-
`tionation of the RNA species during the chromatography
`enables the isolation of the RNA species in low volumes
`due to the low flow rates (3 mL/min) used during the chro-
`matography. Therefore, no further sample manipulation and
`downstream processing of the sample is required, thereby
`significantly reducing potential RNase contamination.
`To demonstrate the ability to enrich and fractionate
`miRNAs from total RNA populations, a total RNA fraction
`was spiked with a synthetic miRNA corresponding to the
`let-7 miRNA sequence (see Materials and Methods). The
`small RNAs were subsequently enriched using solid phase
`extraction as previously described, and the enriched small
`RNAs were analyzed using IP RP mHPLC (see Fig. 4). These
`results demonstrate the ability to efficiently enrich the
`miRNAs and subsequently separate the miRNAs from the
`smaller RNA species.
`
`CONCLUSIONS
`
`Ion pair reverse phase chromatography of RNA under
`denaturing conditions enables the high-resolution separa-
`tion of RNA in relatively short analysis times. We have
`shown that both small RNAs (tRNAs, 5S rRNA) and larger
`rRNA species can be selectively separated during chroma-
`tography. Such approaches could be extended to the detec-
`tion of microorganisms on the basis of their 16S rRNA gene
`sequences. The retention time is likely to be affected by both
`the size and the sequence of the rRNA and may allow the
`discrimination of closely related 16S rRNA genes.
`Recently the focus of much molecular biology research
`has been the roles played by a group of small RNA species
`referred to as small nuclear RNAs (including siRNAs and
`miRNAs). The increasing importance of the identification
`of such miRNAs requires the development of technology to
`overcome many of the problems associated with such meth-
`ods, including the enrichment of small RNAs from total
`RNA extracts. Here we have demonstrated the enrichment
`of small RNAs (tRNA, 5S rRNA) from the larger, more
`abundant rRNA species using solid phase extraction cen-
`tered around ion pair methodology. Using such methodol-
`ogy also allows the enrichment of siRNA and miRNAs from
`total RNA extracts and was demonstrated using synthetic
`RNA corresponding to the let-7 miRNA sequence. Further-
`more the use of solid phase extraction methodology will
`allow the scale up of large quantities of RNA and control
`over the elution parameters to selectively elute the RNAs of
`interest. In addition, capillary chromatography in conjunc-
`tion with ion pair methodology has enabled very sensitive
`approaches to be developed in the analysis of RNA, requir-
`ing only sub-low nanogram quantities of RNA. Using such
`techniques allows the detection of very low levels of RNA
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`RNA analysis centered on IP RP HPLC
`
`Gradient 3: Buffer A, 0.1 M TEAA (pH 7.0) (Fluka); buffer B,
`0.1 M TEAA (pH 7.0) containing 25% acetonitrile. Starting at 25%
`buffer B the gradient was extended to 30% buffer B in 2 min,
`followed by an extension to 50% buffer B over 15 min at a flow
`rate of 3.0 mL/min.
`Gradient 4: Buffer A, 0.1 M TEAA (pH 7.0) (Fluka); buffer B,
`0.1 M TEAA (pH 7.0) containing 25% acetonitrile. Starting at 25%
`buffer B the gradient was extended to 30% buffer B in 2 min,
`followed by an extension to 55% buffer B over 15 min, followed
`by an extension to 60% buffer B over 2 min at a flow rate of 3.0
`mL/min.
`Gradient 5: Buffer A, 0.1 M TEAA (pH 7.0) (Fluka); buffer B,
`0.1 M TEAA (pH 7.0) containing 25% acetonitrile. Starting at 15%
`buffer B the gradient was extended to 20% buffer B in 2 min, followed
`by an extension to 45% buffer B over 15 min, followed by an exten-
`sion to 60% buffer B over 2 min at a flow rate of 3.0 mL/min.
`
`Solid phase extraction and enrichment of small RNAs
`
`Total RNA was extracted from 1 3 106 Hela cells (CILBIOTECH)
`using standard phenol/chloroform extraction procedures. Fol-
`lowing extraction the total RNA was resuspended in 0.1 M TEAA
`(Fluka) (pH 7.4), 10% acetonitrile (HPLC grade, Sigma). Solid
`phase extraction columns were prepared using 8 mm alkylated
`poly(styrene-divinylbenzene) (Transgenomic) particles suspended
`in 50% acetonitrile, 0.2 M TEAA using 5 mL columns (Pierce) The
`columns were equilibrated with two column volumes of 0.2 M
`TEAA in 10% acetonitrile. The total RNA fraction was passed
`down the solid phase extraction column and the unbound fraction
`retained. Subsequently tetrabutylammonium bromide (TBAB)
`(Fluka) was added to the unbound fraction to a final concentra-
`tion of 5 mM. This fraction was then added to a second solid phase
`extraction column that had previously been equilibrated with
`two column volumes of 5 mM TBAB. Both columns were washed
`with five column volumes of the appropriate ion pair solution.
`Elution of the RNA from the columns was performed by the
`addition of 1–2 column volumes of 50% acetonitrile. Let-7 miRNA
`(5¢-UGAGGUAGUAGGUUGUAUUAGUU-3¢) was obtained from
`Sigma Proligo. Twenty micrograms (20 mg) of total RNA was spiked
`with 150 ng of let-7 miRNA. Subsequently the small RNAs were
`enriched using solid phase extraction as previously described.
`
`Received October 28, 2005; accepted December 28, 2005.
`
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`FIGURE 4. IP RP mHPLC chromatogram of the enriched small RNAs
`from total RNA spiked with synthetic let-7 miRNA. The chromato-
`gram shows the analysis of the small RNA fraction obtained following
`the solid phase enrichment of a total RNA (20 mg) spiked with
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`MATERIALS AND METHODS
`
`IP RP HPLC
`
`All samples were analyzed by IP RP HPLC on an Agilent 1100
`HPLC using a DNAsep column 4.6 3 50 mm i.d. (Transgenomic).
`The stationary phase of the column consists of a nonporous,
`alkylated poly(styrene-divinylbenzene) matrix. Chromatograms,
`analyzed using UV detection, were recorded at a wavelength of
`260 nm.
`The IP RP HPLC analysis was performed using the following
`conditions:
`Gradient 1: Buffer A, 0.1 M TEAA (pH 7.0) (Fluka); buffer B,
`0.1 M TEAA (pH 7.0) containing 25% acetonitrile (Sigma). Start-
`ing at 30% buffer B the gradient was extended to 32% buffer B in 2
`min, followed by an extension to 55% buffer B over 15 min at a
`flow rate of 1.0 mL/min.
`Gradient 2: Buffer A, 0.1 M TEAA (pH 7.0) (Fluka); buffer B, 0.1 M
`TEAA (pH 7.0) containing 25% acetonitrile. Starting at 20% buffer B
`the gradient was extended to 22% buffer B in 2 min, followed by an
`extension to 52% buffer B over 15 min, followed by an extension to
`65% buffer B over 2.5 min at a flow rate of 1.0 mL/min.
`
`IP RP mHPLC
`
`All samples were analyzed by IP RP mHPLC on an Ultimate
`capillary HPLC system (LC Packings) using a poly(styrene-di-
`vinylbenzene) monolith column 60 3 0.2 mm i.d. (LC Packings).
`Chromatograms, analyzed using UV detection, were recorded at a
`wavelength of 260 nm using a 45 nL flow cell (LC Packings).
`The IP RP mHPLC analysis was performed using the following
`conditions:
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`Enrichment and analysis of RNA centered on ion pair reverse phase
`methodology
`
`MARK J. DICKMAN and DAVID P. HORNBY
`
` 2006 12: 691-696
`RNA
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