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Comparative analyses of Saccharomyces cerevisiae RNAs using
Agilent RNA 6000 Nano Assay and agarose gel electrophoresis
ic›icova¤, Zdena Palkova¤
Marke¤ta R
Department of Genetics and Microbiology, Charles University, Vinic›na¤ 5, 12844 Prague 2, Czech Republic
Received 13 January 2003; received in revised form 19 April 2003; accepted 22 April 2003
First published online 20 May 2003
Abstract
Precise quantification and quality characterisation of isolated RNAs are prerequisites for their further exploitation in genome-wide
microarrays, Northern blots, cDNA library preparation and others. Our data indicate that RNA analyses using Agilent RNA Nano
Assay exhibit several advantages when compared with those performed on ethidium bromide-stained agarose gel electrophoresis or on a
spectrophotometer. The RNA Nano Assay makes it possible to estimate RNA concentrations in the range from 1000 ng Wl31 to 17 ng
Wl31 . The presence of impurities including traces of DNA within RNA samples does not influence the concentration measurements. Like
agarose gel electrophoresis, RNA Nano Assay allows to analyse RNAs dissolved in formamide and therefore protected against RNase
action. Moreover, it allows a clearer distinction of partially degraded samples. The limitation of RNA Nano Assay is the impossibility to
detect and to analyse double-stranded RNAs.
4 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : RNA analysis; Agilent Bioanalyzer RNA 6000 Nano Assay; Agarose electrophoresis ; Double-stranded RNA
1. Introduction
The sequencing of various genomes including that of the
yeast Saccharomyces cerevisiae has recently resulted in the
development of various new methodical approaches as genome-wide microarrays comparing transcriptomes extracted either from di¡erent yeast strains or from a particular strain growing under di¡erent conditions [1]. A
crucial step of this technique is preparation of large quantities of yeast RNA, its quanti¢cation and quality estimation. Additionally, precise RNA quanti¢cation is even
more important when northern blots, either routinely controlling microarray results or extending the expression
studies on a particular gene, are used. Classical techniques
of RNA separation using slab agarose gels are laborious
and require e⁄cient and permanent protection of RNAs
against the action of RNases [2]. Simple RNA quanti¢cation by spectrophotometry does not precisely distinguish
between intact and partially degraded RNA. Here, we
compare the advantages and disadvantages of yeast
* Corresponding author. Tel. : +420 (2) 21951721;
Fax : +420 (2) 21953316.
E-mail address : [email protected] (Z. Palkova¤).
RNA analyses performed by the classical electrophoretic
technique to those performed on the Agilent Bioanalyzer
using RNA 6000 Nano Assay [3]. We show that RNA
6000 Nano Assay su⁄ciently combines the qualitative
and quantitative evaluation of total yeast RNAs.
2. Materials and methods
2.1. Total yeast RNA extraction
Total yeast RNAs were isolated from S. cerevisiae
BY4742 (MATK, his3v1, leu2v0, lys2v0, ura3v0) (Euroscarf). Brie£y, approximately 109 cells were suspended in
TES bu¡er (10 mM Tris, pH 7.5, 10 mM EDTA, 0.5%
sodium dodecyl sulfate (SDS)) and the RNA was extracted using glass beads and acid phenol/chloroform
treatment (detailed protocol on http://www.biologie.
ens.fr/en/genetiqu/puces/microarraysframe.html) [4]. After
ethanol precipitation, RNA was dissolved either in distilled water or in formamide (as indicated).
2.2. RNA separations
RNA samples were separated either on 1.5% ethidium
1567-1356 / 03 / $22.00 4 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S1567-1356(03)00145-4
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M. R
bromide (EtBr)-stained agarose gels as described [5] or
using Agilent Bioanalyzer 2100 (Agilent Technologies,
Palo Alto, CA, USA) and Agilent RNA Nano Assay
(RNA Nano reagent kit guide, part # G2941-90125).
surement, or (after separation on slab agarose gel) by
comparing samples with reference RNA of known concentration. Both techniques have serious disadvantages. If the
RNA sample contains traces of DNA, phenol or other
UV-absorbing compounds, the spectrophotometer mea-
2.3. Isolation of nucleic acids from S. cerevisiae T158C
log (concentration)
The total yeast nucleic acid material containing doublestranded (ds) RNAs was extracted from stationary-phase
cells of S. cerevisiae T158C (a, his3 ) (Collection of the
Department of Genetics and Microbiology, Charles University, Prague, Czech Republic) using the ‘frost method’
[6]. Brie£y, stationary cells were washed with ethanol,
dried for 30 min and disrupted by mixing in the presence
of liquid nitrogen. The lysate was treated for 1 h with
proteinase K (0.05 mg ml31 ), extracted with phenol/chloroform and precipitated by ethanol. Nucleic acids were
dissolved in TE bu¡er (10 mM Tris^HCl, pH 8, 1 mM
EDTA^NaOH, pH 8.0).
3.5
2016
1044
3
541
315
167
2.5
85
2
1.5
17
11
7
1
2.4. Isolation of the chromosomal DNA from yeast cells
6
5
0.5
0
1
1
3
4
log (dilution)
2
7
6
17
11
20
16
10
44
54
1
31
5
16
7
85
A
5
1
0
M
The chromosomal DNA was extracted from 2 ml of
overnight yeast culture. The yeast cell pellet was suspended in 150 Wl of TEMZ bu¡er (50 mM Tris, 10 mM
EDTA, pH 8, 0.3% L-mercaptoethanol, 1 mg ml31 zymolyase) and incubated for 1 h at 37‡C. Then, 20 Wl of 10%
SDS and 100 Wl of 7.5 M CH3 COONH4 were added and
after 15 min incubation at 380‡C, the sample was centrifuged for 15 min at 15 000 rpm. Supernatant was extracted
twice with one volume of phenol/chloroform (1:1) and
DNA precipitated by 0.6 volume of isopropanol for 15
min at 380‡C. After centrifugation, the DNA pellet was
dissolved in 20 Wl of distilled water.
3. Results and discussion
B
NA
AG
11
17
85
M
Using classical approaches, the concentration of isolated
RNA can be determined either by spectrophotometer mea-
16
10
44
54
1
31
5
16
7
RNA Nano Assay
20
3.1. Quanti¢cation of total RNA isolated from S. cerevisiae
C
Fig. 1. Estimation of RNA concentration using RNA Nano Assay and
EtBr-stained agarose electrophoresis. A: Concentration calibration curve
of diluted RNAs performed on Nano Assay. Numbers within the graph
indicate amounts of analysed RNA in ng. Arrows indicate the range of
concentrations reproducibly determined on Nano Assay. B,C: RNA
samples of di¡erent concentrations separated (B) using Nano Assay
(NA) (scaling performed according to lane 167) or (C) on EtBr-stained
agarose gel (AG) electrophoresis. Numbers indicate RNA concentration
in ng as detected on NA. The same RNA samples were used in A, B
and C. Line 7S of NA (B) was scaled independently to show the intactness of the RNA. D: Yeast chromosomal DNA separated using Nano
Assay (NA) or EtBr-stained agarose gel (AG) electrophoresis. The same
chromosomal DNA was used for NA and AG. RNA-6000 ladder (Ambion 7152) and RNA marker (Promega G3191) were used as RNA
markers in B and C, respectively.
D
C
FEMSYR 1577 23-9-03
EtBr-stained agarose gel
5 ic›icova¤, Z. Palkova¤ / FEMS Yeast Research 4 (2003) 119^122
M. R
121
Fig. 2. Comparison of di¡erent RNA samples using RNA Nano Assay. (A) The same RNA dissolved in either 100% formamide or the distilled water
and separated on NA. (B) Partially degraded (a,c) or intact (b,d) RNA samples separated on NA or AG. Arrows indicate peaks determining degraded
RNAs.
surement can give a wrong estimate of the RNA concentration. Contaminated samples can be more precisely evaluated on agarose gels, in parallel with calibration dilutions
of known RNA. The image of the gel can be subsequently
analysed using densitometer scanning. Nevertheless, this
kind of RNA evaluation is laborious and time-consuming.
The Agilent RNA Nano Assay allows to estimate the
RNA concentration in parallel with RNA separation
and its qualitative analysis. Fig. 1A shows the range of
RNA concentrations that can be determined reproducibly
using RNA Nano Assay. In contrast to spectrophotometer
measurements, where traces of chromosomal DNA within
samples in£uence the rated concentration, the RNA Nano
Assay does not detect high-molecular-mass DNAs (Fig.
1D) and, therefore, calculation of concentrations is not
a¡ected. However, this should be kept in mind in situations when the purity of RNA samples is critical for subsequent applications (e.g. reverse transcription polymerase
chain reaction). In such a case RNA Nano Assay analysis
should be combined with some other technique, which
allows detection of DNA in a sample.
Another advantage of RNA Nano Assay is its sensitivity. We compared the sensitivity of slab agarose gel electrophoresis (Fig. 1C) and RNA Nano Assay separation
(Fig. 1B) using the same RNA samples diluted in formamide. The lowest limit of RNA concentration that can be
detected on EtBr-stained agarose gel is approximately
170 ng, while RNA Nano Assay allows us to detect even
7 ng of RNA in a 24 times more diluted sample (Fig. 1B,
lane 7S). Moreover, whereas the concentration curve obtained from RNA Nano Assay measurement is reproducible and linear in the concentration range from 1000 to
17 ng (Fig. 1A), the EtBr-stained agarose gel does not
allow linear concentration detection when the amount of
separated RNA drops below 500 ng (Fig. 1C). The high
sensitivity of RNA Nano Assay could be very useful when
expression changes within multicellular yeast communities
(colonies or bio¢lms) are analysed. Previously, meaningful
gene expression changes were monitored within distinct
areas of S. cerevisiae colonies using LacZ gene fusions
[7,8]. The opportunity to quantify total RNA extracted
from a limited number of cells picked up from speci¢c
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M. R
3.3. RNA Nano Assay does not detect S. cerevisiae dsRNA
killer toxin determinants
Some yeast strains of di¡erent genera contain in their
cytoplasm dsRNAs, packed within non-infective virus-like
particles [6,9]. In order to ¢nd out if dsRNAs can be
distinguished on RNA Nano Assay, we isolated the total
nucleic acid content of S. cerevisiae strain T158C [6],
which contains two dsRNAs di¡ering in molecular mass
(4.6 kb and 1.8 kb, respectively) [10]. Fig. 3 shows that no
dsRNAs of these molecular masses are detectable using
RNA Nano Assay, in spite of their relatively high concentration within the samples as indicated on EtBr-stained
agarose gel.
Acknowledgements
Fig. 3. Separation of samples containing yeast dsRNAs using RNA
Nano Assay and EtBr-stained agarose electrophoresis. Nucleic acids
were isolated from S. cerevisiae strain T158C as described in Section 2.
areas of a yeast colony for subsequent Northern blot analyses with speci¢c probes could be of considerable interest.
3.2. RNA Nano Assay makes it possible to analyse RNAs
protected against RNases by formamide
When comparative studies on the content of particular
mRNAs within a total yeast RNA are performed, optimally the identical total RNA sample (sample of identical
concentration) should be used for each Northern blot detection. In such a case, repeated manipulations increase
the probability of RNA degradation. Therefore, protection of RNAs (e.g. by formamide) already during their
concentration measurements is advantageous. However,
the presence of formamide excludes the possible exploitation of a spectrophotometer, only agarose gel electrophoresis can be used. For these reasons, we were interested in
the possibility of performing RNA Nano Assay with the
RNA dissolved in formamide. Fig. 2A shows that formamide RNA samples are separated even better than identical samples dissolved in distilled water according to the
original protocol. The pro¢le of rRNAs routinely used for
checking RNA quality is more precise when formamide
RNA samples are used. This indicates that the more e⁄cient dissolving of RNA in formamide helps the separation, as was described in the case of the formamide agarose gels. Additionally, the pro¢le of rRNA peaks readily
allows determination of samples containing partially degraded RNAs (Fig. 2Ba,c). They can be easily distinguished even from those containing an atypical pro¢le of
ribosomal RNAs (Fig. 2Bd), which we detected e.g. in
yeast cells in late stationary phase (unpublished data).
This work was supported by Agilent Technologies and
R 204/02/
by grants from the Czech Grant Agency GAC
0650, and the Grant Agency of Charles University GAUK
141/2001/B-BIO/PrF.
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