Multiparametric duplex real-time nucleic acid sequence-based amplification assay for mRNA profiling.
ABSTRACT Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method. We have developed real-time NASBA assays to detect mRNA coding for the estrogen receptor alpha (ESR1) and the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB) as the normalizing gene. Both the ESR1/PPIB and PGR/PPIB duplex NASBA assays are highly sensitive, specific, and reproducible. Quantification is determined using external standard calibration curves and the ratio between the number of target and housekeeping gene mRNA copies. Amplification of the target gene in the duplex NASBA assay was disrupted when this latter was mixed with a large amount of the housekeeping PPIB gene, suggesting that it is preferable for the normalizing gene chosen to have an expression level comparable to the target gene. Sensitivity and robustness of the duplex NASBA assays were assessed in breast cancer cell lines. Such a rapid and easy-to-use multiparametric duplex real-time NASBA assay could also advantageously be set up for other mRNA profiling applications.
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ABSTRACT: Nucleic acid sequence-based amplification (NASBA) is a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature.Nature 04/1991; 350(6313):91-2. · 38.60 Impact Factor
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ABSTRACT: NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription/polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron spanning primer pairs to control false positive results in RT-PCR. Using NASBA, mRNA of the intronless human interferon-beta gene was demonstrated with a sensitivity of 10 copies, whereas 100 ng genomic DNA gave a negative result.Nucleic Acids Research 06/1998; 26(9):2250-1. · 8.28 Impact Factor
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ABSTRACT: Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA can also be amplified. This amplification system uses a battery of three enzymes (avian myeloblastosis virus reverse transcriptase, RNase H and T7 RNA polymerase) leading to main amplification product of singlestranded RNA. Expensive equipments are not necessary to acquire a high level of precision. NASBA is an established diagnostic tool in clinical use, with a theoretically bigger analytical sensitivity than reverse transcription-polymerase chain reaction (RT-PCR) for pathogen detection. It has a potential for detection of viable cells through selective amplification of messenger RNA, even in a background of genomic DNA, which PCR does not possess. In the future, NASBA could be used to identify and subsequently quantify microorganisms (even those which cannot be readily cultured) and would be very efficient as routine diagnostic procedures.International Journal of Life Science & Pharma Research. 03/2012; 2(1-ISSN 2250-0480):106-121.
476 BioTechniques Vol. 37, No. 3 (2004)
PRODUCT APPLICATION FOCUS
Gene expression monitoring is becoming increasingly
popular in the biomedical research community. This ap-
proach requires a reliable amplification method that allows
even small amounts of input RNA to be quantified. Nucleic
acid sequence-based amplification (NASBA) has been de-
scribed as an efficient method of amplifying RNA at 41°C
using T7 RNA polymerase, RNase H, avian myeloblastosis
virus (AMV) reverse transcriptase, and two specific primers
(1–3). NASBA has been successfully approved for the de-
tection of various mRNAs (4) and viral and bacterial RNA
in clinical samples (5–9). An elegant method of detecting
NASBA products is the use of fluorescent molecular beacons
(10), which enable homogeneous real-time detection of RNA
To determine the quantity of target nucleic acids in the
sample, they are quantified using external calibration curves.
We have developed real-time NASBA-based assays using du-
plex analysis of the gene of interest and a housekeeping gene.
This duplex approach could be employed to monitor several
target genes in parallel, thereby providing a convenient tool
for multiparametric mRNA profiling.
In the present study, we describe the development of two
duplex real-time NASBA assays to investigate, in breast
tumors, level of mRNA coding for the estrogen receptor α
(ESR1) and the progesterone receptor (PGR) with the cy-
clophilin B (PPIB) housekeeping gene, whose expression has
been shown to be stable in breast cancer cell lines and tumors
MATERIALS AND METHODS
Cell Line Culture and Total RNA Extraction
Two tumor cell lines were used in the study, breast carcino-
ma MCF-7 and BT-549 cells. Both cell lines were purchased
from ATCC (Manassas, VA USA). The MCF-7 cell line was
grown in Dulbecco’s modified Eagle medium (DMEM; Invi-
trogen Canada, Burlington, ONT, Canada), while the BT-549
cells were grown in RPMI 1640 (Invitrogen Canada). Both
media were supplemented with 10% fetal bovine serum (In-
vitrogen Canada), 2 mM L-glutamine (Sigma-Aldrich, Lyon,
France), 1% nonessential amino acids (Invitrogen Canada),
and 10 μg/mL streptomycin (Invitrogen Canada) at 37°C in a
5% CO2 atmosphere. Total RNA was purified from cell lines
using TRIzol® reagent as recommended by the manufacturer
(Invitrogen Canada). RNA quality and quantity were deter-
mined using RNA 6000 Nano LabChips® (Agilent Technolo-
gies, Waldbronn, Germany). RNA samples were stored at
-70°C until use.
Multiparametric duplex real-time nucleic
acid sequence-based amplification assay for
Thibault Verjat, Elisabeth Cerrato, Marcel Jacobs, Philippe Leissner, and Bruno Mougin
BioTechniques 37:476-481 (September 2004)
Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method. We have
developed real-time NASBA assays to detect mRNA coding for the estrogen receptor α (ESR1) and the progesterone receptor (PGR) in
breast tumors by means of duplex reactions using cyclophilin B (PPIB) as the normalizing gene. Both the ESR1/PPIB and PGR/PPIB
duplex NASBA assays are highly sensitive, specific, and reproducible. Quantification is determined using external standard calibra-
tion curves and the ratio between the number of target and housekeeping gene mRNA copies. Amplification of the target gene in the
duplex NASBA assay was disrupted when this latter was mixed with a large amount of the housekeeping PPIB gene, suggesting that
it is preferable for the normalizing gene chosen to have an expression level comparable to the target gene. Sensitivity and robustness
of the duplex NASBA assays were assessed in breast cancer cell lines. Such a rapid and easy-to-use multiparametric duplex real-time
NASBA assay could also advantageously be set up for other mRNA profiling applications.
bioMérieux, Marcy l’Etoile, France
Vol. 37, No. 3 (2004) BioTechniques 477
NASBA quantification was based on a standard curve
with a known input of RNA transcribed from plasmids. These
plasmids were generated by cloning a specific PCR prod-
uct for each target gene. For ESR1 (GenBank® accession
no. X03635), forward primer (5′-TACAGGCCAAATTCA-
GATAATCGAC-3′) and reverse primer (5′-GGAACCGA-
GATGATGTAGCCA-3′) were located at positions 808 and
1666, respectively, generating a 858-bp fragment. For PGR
(GenBank accession no. NM_000926), forward primer (5′-
TGACAAGTCTTAATCAACTAGG-3′) and reverse primer
(5′-TCACTTTTTATGAAAGAGAAGGG-3′) were located at
positions 2319 and 2977, respectively, generating a 658-bp
fragment. For PPIB (GenBank accession no. M60857), for-
ward primer (5′-acatgaaggtgctccttgcc-3′) and reverse primer
(5′-GTCCCTGTGCCCTACTCCTT-3′) were located at posi-
tions 11 and 650, respectively, generating a 639-bp fragment.
Each PCR fragment was cloned into the pGEM-T plasmid
(Promega, Madison, WI, USA) and verified by sequencing.
In vitro transcription was generated from these plasmids us-
ing T7 RNA polymerase (MEGAscript™ Kit; Ambion, Aus-
tin, TX, USA) and treated with DNase to remove the plasmid.
RNA products were purified using the RNeasy® Mini Kit
(Qiagen, Hilden, Germany). Integrity and quantity of RNA
were evaluated with the RNA 6000 Nano LabChips by the
nature of the RNA 18S- and 28S-peaks and the concentration
in ng/mL respectively. The calculation of the RNA copy num-
ber can then be performed based on the concentration and the
length of the NASBA product. The resulting RNAs were used
to set up the ESR1-, PGR-, and the PPIB-standard curves.
Duplex Real-Time NASBA
Real-time detection of NASBA products was performed
with the NucliSens EasyQ® Analyzer (bioMérieux bv, Box-
tel, The Netherlands) using molecular beacons, which are
DNA oligonucleotides labeled with the fluorophore FAM
(6-carboxy-fluorescein) or ROX (6-carboxy-X-rhodamine) at
the 5′ end and a quencher (Dabsyl) at the 3′ end. Probes were
synthesized by Eurogentec (Seraing, Belgium). Molecular
beacons hybridized to the products during amplification and
emitted a fluorescence signal that was measured every 45 s,
thereby allowing real-time detection.
Duplex NASBA was performed using the NucliSens Basic
Kit (bioMérieux bv). Briefly, 5 μL (5 ng) RNA were added
to 10 μL NASBA buffer [final concentration in 20 μL reac-
tion mixture: 40 mM Tris-HCl, pH 8.5, 12 mM MgCl2, 70
mM KCl, 5 mM dithiothreitol, 15% v/v dimethylsulfoxide
(DMSO), 1 mM of each dNTP, 2 mM of each NTP]. For
ESR1/PPIB duplex NASBA, 0.2 μM ESR1 primers, 0.2 μM
PPIB primers, and 0.1 μM of each cognate molecular bea-
con were added, while for the PGR/PPIB duplex NASBA, 0.1
μM PGR primers, 0.2 μM PPIB primers, and 0.1 μM of each
cognate molecular beacon were added. The mixture was then
pre-incubated at 65°C for 2 min, followed by 2 min at 41°C.
Five microliters of enzyme mixture (0.08 U RNase H, 32 U
T7 RNA polymerase, 6.4 U reverse transcriptase) were then
added to start the RNA amplification and incubated at 41°C
for 90 min. Primer sets used as well as molecular beacon se-
quences are shown in Table 1.
RNA Quantification in the NASBA Assay
The time necessary to generate a fluorescent signal that
rises above the threshold of detection and becomes positive
is termed time-to-positivity (TTP). Using NucliSens EasyQ
Analysis software (bioMérieux bv), the signal positivity
threshold was determined, as described previously (12,13), by
setting the fluorescence emitted in the first five measurement
points as a background. Several duplex calibration curves
were drawn using either 10-fold serial dilutions of RNA tran-
scripts (108–102 copies) of the gene of interest and the house-
keeping gene or using 108–102 copies of RNA transcripts of
the target gene mixed with a constant quantity (107 or 105 or
103 copies) of the housekeeping gene. Thus, for each experi-
ment, the number of mRNA copies per sample input could
be extrapolated from those standard curves. Quantification of
Table 1. Sequences of Primers and Molecular Beacons Used for the Detection of ESR1, PGR, and PPIB mRNA
ESR1 P2 1427–-1447
PGR P2 2761–-2780
PPIB P1(T7) 449–-470
Molecular BeaconSequence Position
The 3′ antisense primers are elongated with a T7 promotor recognition sequence indicated in lowercase characters. The stem se-
quences of the beacons are indicated in lowercase italic characters. The positions are in reference to the GenBank accession nos.
X03635 for ESR1, NM_000926 for PGR, and M60857 for PPIB, respectively. ESR1, estrogen receptor α; the progesterone receptor
(PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB).
478 BioTechniques Vol. 37, No. 3 (2004)
PRODUCT APPLICATION FOCUS
the target gene expression was expressed as the mRNA copy
number per 5 ng of total RNA.
RESULTS AND DISCUSSION
Specificity and Sensitivity of Duplex ESR1/PPIB and
PGR/PPIB Real-Time NASBA with RNA Transcripts
The specificity of the NASBA assay was strengthened by
triple hybridization of the target RNA with two specific prim-
ers and a specific detection probe. The specificity of the as-
say was tested by mixing the ESR1 molecular beacon with
106 PGR RNA molecules and vice versa. In both cases, no
signal was observed (data not shown). In addition (see Fig-
ure 3), no fluorescence signal was observed when NASBA
was performed with total RNA extracted from BT-549 cells
known to be estrogen receptor α-negative and progesterone
receptor-negative when assessed by the ligand binding assay
(data not shown), confirming that molecular beacons do not
interact with nonspecific targets.
To evaluate the efficacy of duplex NASBA, amplification
curves of a 10-fold serial dilution of in vitro-synthesized RNA
were plotted. The input of RNA transcripts was 102–108 mol-
ecules for both genes in the same tube. In each such “equimo-
lar” duplex NASBA reaction, molecular beacons specific to
ESR1 and PPIB and to PGR and PPIB mRNA were added.
As shown in Figure 1, the TTP was linear over a range of
at least six orders of magnitude of input RNA molecules.
In the ESR1/PPIB duplex NASBA, the linear quantification
of ESR1 was determined to be between 102 and 108 copies
of RNA per reaction (Figure 1A) and between 103 and 108
copies per reaction for PPIB (Figure 1B). The limit of detec-
tion (LOD) determined with the amplification plots (data not
shown) as well as the limit of quantification (LOQ) at which
accurate TTP values can be calculated could be determined.
The LOD and the LOQ for ESR1/PPIB duplex could be deter-
mined at 102 copies and 103 copies, respectively. In the PGR/
PPIB duplex NASBA, the linear quantification of both mark-
ers was determined to be between 102 and 108 copies of RNA
per reaction (Figures 1C and 1D). The LOD was observed at
102 copies for PGR and PPIB, while the LOQ was reached
at 102 and 103 copies for PGR and PPIB, respectively. These
results illustrate that two different genes can be amplified and
detected simultaneously in real-time with NASBA.
Dynamic Range of the Duplex NASBA
In order to assess a putative influence of the quantity of
PPIB on the dynamic range of ESR1 and PGR amplification
in duplex NASBA experiments, we mixed 102–108 transcripts
of ESR1 or PGR with an equimolar amount of PPIB gene. A
serial dilution of target RNA transcripts (102–108 copies) was
also co-amplified with a constant concentration (103 or 105
or 107 copies) of the housekeeping gene. As shown in Fig-
ure 2, when a low copy number (102–105) of the ESR1 gene
was mixed with a high copy number (105 or 107) of PPIB, the
amplification efficacy of the target gene decreased. Similar
Figure 1. ESR1/PPIB and PGR/PPIB duplex NASBA standard curves with in vitro synthesized RNA. Relationship of time-to-positivity (TTP) to (A)
ESR1, (B and D) PPIB, and (C) PGR RNA copy number. The input of RNA copies present in the reaction is indicated on the logarithmic x-axis while the TTP
value (in min) is indicated on the ordinate. The TTP values are the mean (± sem) of eight independent experiments. NASBA, nucleic acid sequence-based am-
plification; ESR1, estrogen receptor α; the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB).
Vol. 37, No. 3 (2004)
results were observed with PGR in a PGR/PPIB duplex NAS-
BA reaction (data not shown). These results demonstrate that,
in duplex NASBA, the amplification efficacy of a given gene
can be influenced by high numbers of a second gene present
in the reaction. This finding, which has also been described
for reverse transcription PCR (RT-PCR) (14,15), suggests
that the housekeeping gene should have a gene expression
level comparable to that of the target gene. Thus, care should
be taken to select the best normalized gene for each target in a
given tissue. Another way to minimize the variation generated
by multiplex amplification would be to optimize the primer
concentration to achieve efficient amplification for each gene.
An internal calibrator RNA has also proven useful for quanti-
fying gene expression, but this system requires time-consum-
ing optimization of primer concentrations (16,17). However,
while accurate quantification is necessary for various appli-
cations, such as infectious diseases, our objective here is to
propose relative quantification of hormone receptor RNA as
a marker in correlation with the clinical features of the tumor.
Thus, rather than calculating an absolute number of ESR1
and PGR copies, the main issue will be to determine a gene
expression cut-off strongly associated with the presence of
receptors in the cytosol, beyond which hormonotherapy could
be proposed to the patient.
Measurement of ESR1 and PGR mRNA Levels by Duplex
Real-Time NASBA with Total RNA from Cell Lines
In order to evaluate the performance of ESR1/PPIB and
PGR/PPIB duplex NASBA from small quantities of total
RNA, 5 ng extracted from two breast cancer cell lines were
used as starting material. MCF-7 cells were shown to be es-
trogen receptor α-positive/progesterone receptor-positive
and BT549 cells to be estrogen receptor α-negative/proges-
terone receptor-negative by immunohistochemistry (ATCC).
This data was confirmed by a ligand binding assay that mea-
sures the content of functional cytosolic receptors (data not
ESR1, PGR, and PPIB genes were amplified and quanti-
tatively detected using an equimolar duplex standard curve.
Both ESR1 and PGR messengers were found in MCF-7 cells.
In contrast, messengers seemed to be weakly expressed (be-
low the LOD) or not at all in the BT-549 cells (Figure 3).
The internal control PPIB was found to be positive and gene
expression level similar in both cell lines. Quantitative RT-
PCR analysis of ESR1 expression was performed and con-
firmed the NASBA results (data not shown). Therefore, rela-
tive quantification of ESR1 and PGR gene expression can be
performed by determining the ratio between the number of
mRNA molecules of the gene of interest and the housekeep-
ing gene (Table 2). These results demonstrate that duplex
real-time NASBA is a suitable technology for simultaneous-
ly monitoring expression of ESR1 and PGR genes in a very
small amount of total RNA.
In conclusion, we are able to amplify and to detect ESR1
and PGR mRNA by decreasing the starting amount of to-
tal RNA to 5 ng, thereby confirming the high amplification
power of duplex NASBA. This efficiency in nucleic acid am-
plification makes the NASBA technique a suitable tool for
480 BioTechniques Vol. 37, No. 3 (2004)
PRODUCT APPLICATION FOCUS
analysis of small clinical biopsies.
The decreasing size of surgical
tumor samples should progres-
sively restrict the use of ligand
binding assays and biochemical
techniques. Along with immuno-
histochemistry, molecular biol-
ogy-based methods can provide
the clinicians with relevant in-
formation from very small clini-
cal samples, such as biopsies and
fine needle aspiration. Finally,
using the eight-tube strip format developed for
the NucliSens EasyQ platform, rapid, one-shot,
easy-to-use, multiparametric, real-time NASBA
assays could also advantageously be set up for
any mRNA profiling applications.
We thank Dr. Pierre-Jean Lamy at the CRLC
Val d’Aurelle in Montpellier for performing the
ligand binding analysis on cell lines and B. Dei-
man for her helpful input.
COMPETING INTERESTS STATEMENT
The authors declare no conflicts of interest.
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Figure 2. Dynamic range of ESR1/PPIB duplex NASBA. Several calibration curves were
compared. Copies of ESR1 RNA transcripts (102–108) were mixed with an equimolar quantity
of PPIB RNA transcripts and co-amplified (cross). In parallel, a serial dilution of ESR1 RNA
transcripts (102–108 copies) was co-amplified with a constant concentration [107 (open square)
or 105 (black diamond) or 103 copies (open triangle)] of the housekeeping gene. The input of
ESR1 RNA copies present in the reaction is indicated on the logarithmic x-axis, and the TTP
values (in min) corresponding to ESR1 amplification efficacy is indicated on the ordinate. The
ESR1 TTP values are the mean (± sem) of three independent experiments. NASBA, nucleic
acid sequence-based amplification; ESR1, estrogen receptor α; the progesterone receptor
(PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB).
Table 2. Ratio of ESR1/PPIB and PGR/PPIB mRNA Copy Number in Breast Cancer Cell Lines by
Duplex NASBA Assay
ESR1/PPIB Duplex NASBAPGR/PPIB Duplex NASBA
2.24 × 104
7.43 × 105
4.43 × 106
3.01 × 10-2
5.42 × 102
2.54 × 106
2.13 × 10-4
—, copy number below the limit of detection; NASBA, nucleic acid sequence-based amplification; ESR1, estro-
gen receptor α; the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin
Figure 3. ESR1, PGR, and PPIB mRNA level in breast cancer cell lines. ESR1/PPIB and
PGR/PPIB duplex NASBA were performed on 5 ng of total RNA from MCF-7 and BT-549
breast cancer cell lines. Through the external equimolar duplex calibration curve depicted in
Figure 2, quantification of ESR1 mRNA (black bars), PGR mRNA (grey bars), and house-
keeping PPIB mRNA (white bars) were calculated. Results are the means (± sem) of three
independent analysis. NASBA, nucleic acid sequence-based amplification; ESR1, estrogen re-
ceptor α; the progesterone receptor (PGR) in breast tumors by means of duplex reactions using
cyclophilin B (PPIB).