Clinical Chemistry 45: 619-624, 1999;
(Clinical Chemistry. 1999;45:619-624.)
© 1999 American Association for Clinical Chemistry, Inc.
Reverse Transcription-Competitive Multiplex PCR Improves Quantification of mRNA in Clinical Samples—Application to the Low Abundance CFTR mRNA
Stefan M. Loitsch1,
Stefan Kippenberger2,
Nurlan Dauletbaev1,
Thomas O.F. Wagner1 and
Joachim Bargon1,a
1
Department of Internal Medicine, Division of Pulmonary Medicine, and
2
Department of Dermatology, University Hospital Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt, Germany.
a Author for correspondence. Fax 0049-69-63017391; e-mail Bargon{at}t-online.de
 |
Abstract
|
|---|
Background: To monitor gene therapy, we wished to quantify cystic
fibrosis transmembrane conductance regulator (CFTR)
mRNA. We developed a PCR-based method to measure
CFTR mRNA in clinical samples.
Methods: Expression was determined by reverse
transcription-competitive multiplex PCR (RCMP) for
CFTR and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) transcripts, and for serial dilutions of two
internal cDNA standards consisting of CFTR and
GAPDH mutants containing short deletions. The RCMP used
simultaneous amplification of the gene of interest with a reporter gene
in one reaction tube. The expression of CFTR was
calculated with reference to the amount of GAPDH to
correct for variations in initial RNA loading.
Results: Amplification of cDNAs derived from different amounts of
RNA (1–4 µg) gave similar GAPDH/CFTR
ratios, with a coefficient of variation (CV) below 7.5%. RCMP was
applied on nasal and bronchial brushings and shows a high variability
of CFTR expression in non-cystic fibrosis donors.
Conclusion: This method is precise and reproducible and
advantageous for use with limited amounts of tissue, such as from
biopsies or from nasal or bronchial brushings.© 1999 American
Association for Clinical Chemistry
|
Introduction
|
|---|
Quantifying cystic fibrosis transmembrane conductance regulator
(CFTR)1
mRNA in vivo is a prerequisite for monitoring gene therapy and
is essential for the understanding of regulation and function of this
gene. However, quantification is difficult, primarily for two reasons.
First, CFTR mRNA is a low abundance mRNA, with ~0.1–1
copies per cell in the respiratory tract (1). Second, the
cellular yield of biopsies and bronchial or nasal brushings is very
low; therefore, Northern blot analysis or the ribonuclease protection
assay are impractical because of their limited sensitivity. Reverse
transcription (RT)-PCR has been shown to be several orders of magnitude
more sensitive than traditional techniques (2), and it is
one of the most widely used approaches for the quantification of mRNA.
Despite the development of a variety of procedures, quantification by
RT-PCR is difficult, and many investigators are skeptical about the
quantitative aspect of PCR (3).
One of the first attempts to use PCR for quantification of
CFTR was the measurement of the relative amount of
CFTR mRNA in a sample by reference to the amount of a
control gene, either in the same (internal standardization) or in a
parallel reaction tube (external standardization) (1).
Because the primers for the different genes commonly have different
annealing kinetics, it is not possible to directly relate the amount of
amplification products of the different genes. Another major
disadvantage of this approach is that quantification is restricted to
the early exponential phase of PCR, where only the template is
limiting. As a consequence, high- and low-copy mRNAs are not suitable
for coamplification, because a high-copy target may already be in the
plateau phase even when a low-copy target is still not detectable. This
could be circumvented in part by the so-called primer dropping method,
in which the primers of the high-copy mRNA were added after some cycles
of the PCR, according to the abundance of the corresponding PCR
products (4). One problem persists in all these approaches:
differences in the initial loading with RNA could drive the PCR in the
plateau phase for the high-copy gene without any control for the
investigator. All these problems mean that such experiments are
imprecise and increasingly inconvenient in practice.
During the last few years, attempts have been made to develop
procedures that drive the PCR reaction to the plateau phase or further,
yielding greater accumulation of product combined with higher
sensitivity and better reproducibility. This was achieved by
competitive PCR (1)(5)(6), which has
been used for the quantification of CFTR mRNA in endometrium
(7). Competitive PCR is based on the competitive
coamplification of a specific target sequence together with known
concentrations of an internal standard (competitor) in one reaction
tube. A dilution series is used either from the analyzed target or the
competitor fragment, and identical amounts of the other component are
added to each of the reaction tubes. The internal standards share
identical primer recognition sites with the target and should be
amplified with the same efficiency. Internal standards using the same
primers for amplification must be different from the target sequence to
allow a physical separation during gel electrophoresis. This is
achieved by modifications such as deletions, insertions, or
additionally introduced restriction sites. However, even competitive
PCR does not control for the initial loading of RNA.
The accuracy of competitive RT-PCR was potentiated by the simultaneous
determination of a gene of interest with a reporter gene in one
reaction tube as a combination of endogenous and exogenous internal
standards (8)(9)(10). The expression of the gene of interest is
then calculated with reference to the titrated concentration of the
reporter gene. We have used this method to investigate small amounts of
material such as nasal or bronchial brushings as well as cell culture.
RT-competitive multiplex PCR improves competitive RT-PCR and mRNA
quantification in general because of the simultaneous amplification of
high- and low-copy mRNA.
|
Materials and Methods
|
|---|
tissues and cell lines
We studied 16HBE14o cells (11) and nondiseased
respiratory epithelium obtained from second-order bronchi by fiberoptic
bronchoscopy with a standard cytology brush or from nasal brushings of
the inferior turbinate with Curaprox LS brushes (Curaden AG).
Cells were suspended immediately in cold DMEM/Ham's F12.
isolation of rna
Total cellular RNA was isolated from nasal and bronchial brushings
by RNAzol BTM (Wak-Chemie). After the addition of
RNAzol B, bronchial epithelial cells were homogenized by
QIAshredderTM (Qiagen). Contaminating DNA was
removed by incubation of 5 µg of total RNA with 1 U of RNase-free
DNase (Boehringer Mannheim) in 50 µL of 1x transcription buffer (50
mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L
MgCl2, 10 mmol/L dithiothreitol; Life
Technologies) for 20 min at 37 °C. The RNA was phenol/chloroform
extracted, isopropanol precipitated, and reconstituted in
diethylpyrocarbonate-treated water.
reverse transcription
RNA (1–5 µg) was reverse transcribed into cDNA in 20 µL of
1x transcription buffer containing 1.5 mmol/L each dNTP, 100–500 ng
of random hexamers, and 200 U of Superscript II-reverse transcriptase
(Life Technologies) in the reaction volume. Incubation was performed
according to the manufacturer's instructions.
construction of internal standards (competitors)
Internal standards (competitors) for CFTR and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were
wild-type fragments containing a deletion, but sharing identical primer
recognition sites with the wild-type target. The fragments were
produced according to the method of Celi et al. (12). As
shown in Fig. 1
, PCR was performed with a conventional ~20-nucleotide PCR
primer (primer A) and a 40-nucleotide primer construct (primer
construct CB). The primer construct CB contains two 20-nucleotide
sequences. The 5' end sequence (B) corresponds to the opposite strand
of the target sequence. The 3' end sequence (C) determines the length
of the PCR product and corresponds to the opposite strand of the target
sequence at a predetermined distance to sequence B. Amplification with
primer A and primer construct CB leads to a PCR product with a deletion
of the nucleotides between sequence C and B. The competitors were
placed in T-vectors (Promega) named pCFIST and pGAPIST. The copy number
was determined after spectrophotometric quantification. For PCR, the
competitors were used as intact plasmids. The wild-type sequences and
the competitors for CFTR and GAPDH were amplified
with Taq Polymerase (Life Technologies) according to the
manufacturer's instructions. The cycling conditions were as follows: a
hot start, followed by 30 cycles of 94 °C for 10 s, 58 °C
for 30 s, and 72 °C for 60 s. Two to four microliters of
cDNA were used in a total volume of 50 µL containing 15 pmol of
primers. The CFTR primers were constructed according to
Trapnell et al. (1). The nucleotide sequences were as
follows: CFTR-A, 5'-ATT ATG GGA GAA CTG GAG CCT-3'; CFTR-B, 5'-GCC ATC
AGT TTA CAG ACA CAG-3'; CFTR-CB, 5'-GCC ATC AGT TAA CAG ACA CAG GAC CTC
CAC TCA GTG TGA TTC-3'. Primer combination CFTR-A/B amplified a 377-bp
product, whereas the combination CFTR-A/CB generated a competitor of
265 bp. The GAPDH primers (GenBank, accession no. M33197) had
the following sequences: GAPDH-A, 5'-ATC TTC CAG GAG CGA GAT CC-3';
GAPDH-B, 5'-ACC ACT GAC ACG TTG GCA GT-3'; GAPDH-CB, 5'-ACC ACT GAC ACG
TTG GCA GTA GTA GAG GCA GGG ATG ATG-3'. Primer combination GAPDH-A/B
amplified a 502-bp product, whereas the combination GAPDH-A/CB
generated a competitor of 427 bp.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 1. Construction of internal standards as competitors for
competitive PCR.
Competitors were constructed according to the method of Celi et al.
(12). (A), reamplification of wild-type cDNA
with primer A and primer CB generates a competitor (B)
with a deletion of the nucleotides between sequences C and B;
(C), reamplification with primer A and primer B.
|
|
amplification kinetics of wild-type and competitor sequences
Essential for the proper performance of quantitative competitive
PCR is an identical or at least comparable amplification efficiency of
competitor and wild-type templates. This was checked by comparison of
the amplification of wild-type sequences with their competitors as a
function of cycle number.
rt-competitive multiplex pcr
The PCR conditions were standardized for each experiment by use of
a master mixture containing 50 mmol/L KCl, 10 mmol/L Tris-HCl, 1.5
mmol/L MgCl2, and 0.5 mmol/L of each dNTP, with
30 pmol of CFTR primer, 5 pmol of GAPDH primer,
and 2 µL of cDNA per 25 µL of total PCR volume. Aliquots of this
master mix were added to serial dilutions (1:3) of a mixture of the
competitors pCFIST (2 x 105 molecules/µL)
and pGAPIST (4 x 101
molecules/µL).
PCR was performed as described above, with the exception that 40 cycles
were used and the extension time was increased to 2 min. Each sample
(10 µL) was electrophoresed in 2% agarose gels. Quantification of
ethidium bromide-stained gels was performed with the DocuGel IV-System
(MWG-Biotech). The ratio of target to competitor was determined,
extrapolated from each point of the curve, and plotted against the
amount of competitor added. As a consequence, the number of molecules
of competitor corresponding to a 1:1 ratio is equivalent to the number
of molecules of input target mRNA.
|
Results and Discussion
|
|---|
To determine the mRNA content of CFTR in bronchial and
nasal brushings, we developed an RT-competitive multiplex PCR in which
CFTR and GAPDH were coamplified with specific
competitors. GAPDH serves as a control for RNA loading
because competitive PCR alone does not control for variation in the
starting amounts of template. Because the ratio of competitor and
target is maintained even after the exponential phase and during the
plateau phase (13)(14), GAPDH can be
coamplified as a high-copy mRNA along with CFTR as a
low-copy mRNA, with 0.1–1 copies/cell in human airways (1).
The primers for GAPDH and CFTR spanned several
exons to prevent amplification products of contaminating DNA.
Amplification with genomic DNA or RNA that had not been transcribed
gave no amplification products (data not shown). The competitors were
then generated by the method described by Celi et al. (12).
Important for the proper performance of competitive PCR is that the
competitor and target are amplified with the same efficiency. Even when
the same primers are used for both the competitor and the target, there
is an inverse relationship between the length of an amplified sequence
and the extent of amplification (15)(16). The
amplification kinetics for CFTR and pCFIST and
GAPDH and pGAPIST were determined by coamplification of the
competitor and the target in equal amounts for various cycle numbers.
Fig. 2
shows an example for CFTR and pCFIST. In both cases,
the competitor and target were amplified with the same kinetics.
However, GAPDH and CFTR can have different
amplification kinetics. Differences in the amplification kinetics of
the different primer sets are eliminated by titration against their
respective competitors.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 2. Amplification kinetics for CFTR and
competitor pCFIST as a function of cycle number.
Amplification products were separated on agarose gels and stained with
ethidium bromide. Band intensities were determined by use of a computer
imaging system.
|
|
Because of the influence of amplification efficiency and initial
abundance, there will be differences in the amount of PCR products
between the different wild-type/competitor systems. This was
circumvented by adjustment of the respective primer concentrations
(GAPDH, 5 pmol/primer; CFTR, 30 pmol/primer),
which influenced the respective plateau phases. A typical
RT-competitive multiplex PCR experiment is shown in Figs. 3
and
4. Lower primer concentrations produce lower amounts of PCR
products for a complete wild-type/competitor system. This is possible
because in competitive PCR the starting ratio of wild-type/competitor
is preserved throughout the entire amplification process. Provided that
no other factor is limiting, the plateau phase of every
wild-type/competitor system can be controlled by the primer
concentrations. This solves two major problems of conventional RT-PCR:
first, high- and low-copy mRNAs can be coamplified; and second, proper
quantification is independent of the differing amplification
efficiencies between the two genes.
View larger version (43K):
[in this window]
[in a new window]
|
Figure 3. Agarose gel with ethidium bromide-stained marker
(M) and CFTR and GAPDH
products of an RT-competitive multiplex PCR assay.
Serial dilutions (1:3) of competitor cDNAs were coamplified
with constant amounts of reverse transcribed total RNA from 16HBE14o
cells, separated on 2% agarose gels, and stained with ethidium
bromide. Comp, competitor.
|
|
Another critical point in competitive PCR is the densitometric
quantification of the ethidium bromide-stained amplification products.
Longer products will give stronger signals than products with deleted
sequences (8). The densitometric values should,
therefore, be corrected for the size differences of competitor and
target, thereby correcting the equivalence points. This is not
necessary in RT-competitive multiplex PCR because the correction factor
remains constant in all determinations so that comparisons of
GAPDH/CFTR ratios between different samples are
not influenced. Furthermore, ethidium bromide staining influences the
titration curve of the competitor/wild-type ratio. Depending on the
ethidium bromide background, the slope will change, but the
equivalence point remains unchanged (data not shown; 9).
In theory, the ratio between GAPDH and CFTR is
independent of the initial amount of RNA. To test this hypothesis, 18
determinations from the same RNA preparation of 16HBE140 cells
(11) were performed, 6 reverse transcription reactions with
4 µg of RNA, 6 reactions with 2 µg of RNA, and 6 reactions with 1
µg of RNA. Our results (Table 1
) demonstrate that coamplification with a housekeeping gene
corrects for variations in initial RNA loading. This aspect is of great
importance when limited samples such as small biopsies, nasal or
bronchial brushings are used. The RNA yields in all of theses samples
can be so low that RNA quantification is not possible. RT-competitive
multiplex PCR gives accurate results even when the exact RNA
concentration is not known.
These results also indicate that cDNA internal standards are suitable
for RT-competitive multiplex PCR. Some authors use cDNA fragments as
competitors (RT-competitive PCR) added after the reverse transcription
step (17), whereas others propose the use of cRNA
competitors (2)(5), added during RNA isolation
or the reverse transcription step to standardize for variations in
isolation or reverse transcription (competitive RT-PCR). Reverse
transcription is an important source of variability because its
efficiency ranges from 5% to 90% (18)(19). In
noncompetitive RT-PCR, endogenous or exogenous mRNA targets compensate
for this high variability [for a review, see Ref. (20)].
This feature was used in RT-competitive multiplex PCR, where
GAPDH functions as a control for the efficiency of the
reverse transcription. However, RT-competitive multiplex PCR with cDNA
internal standards is a semiquantitative approach, whereas the use of
cRNA internal standards added during RNA isolation can be used for
absolute quantification.
RT-competitive multiplex PCR is suitable for the quantification of RNA
in cell culture and in clinical samples. Table 2
shows that the expression of CFTR in
bronchial or nasal brushings of non-cystic fibrosis donors is highly
variable. RT-competitive multiplex PCR improves and simplifies
quantification of gene expression for several reasons: (a)
gene expression can be quantified in very small samples of cells,
giving precise and reproducible results with even fewer than 100
transcripts per PCR reaction, provided that the same competitor-master
mixture is used; (b) quantification is independent of the
starting amounts of total RNA or cDNA because of simultaneous
amplification of a gene of interest (CFTR) with a reporter
gene (GAPDH) in the same reaction tube; (c)
tube-to-tube variability is reduced; (d) the use of cDNA
internal standards instead of cRNA internal standards is sufficient for
semiquantitative purposes and make quantitative PCR much easier to
perform; and (e) titration against internal standards gives
a superior accuracy over Northern blotting and ribonuclease protection
assays. RT-competitive multiplex PCR improves competitive RT-PCR and
combines an increase in sensitivity with a potentiation of accuracy
when compared with other conventional approaches. Furthermore, it is
easy to perform and can be established in every laboratory with
standard molecular biology equipment.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 4. Ratio of target to competitor plotted against
the amount of competitor added for CFTR and competitor
pCFIST (A) and GAPDH and pGAPIST
(B).
The amount of competitor corresponding to a 1:1 ratio is
equivalent to the amount of target mRNA.
|
|
|
Acknowledgments
|
|---|
This work was supported by Bundesministerium für
Bildung und Forschung grant FKZ:01KV9552/6. We thank A. Sewell
for critical reading the manuscript, U. Langenbeck and J. Stein for
financial support, and D.C. Gruenert, Cardiovascular Research
Institute, University of California-San Franscisco, San Francisco, CA,
for the gift of 16HBE14o cells.
|
Footnotes
|
|---|
1 Nonstandard abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; RT-PCR, reverse transcription-PCR; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 
|
References
|
|---|
-
Trapnell BC, Chu C-S, Paakko PK, Banks TC, Yoshimura K, Ferrans VJ, et al. Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis. Proc Natl Acad Sci U S A 1991;88:6565-6569.
[Abstract/Free Full Text]
-
Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A 1989;86:9717-9721.
[Abstract/Free Full Text]
-
Hengen PN. Methods and reagents. Quantitative PCR: an accurate measure of mRNA?. Trends Biochem Sci 1995;20:476-477.
[ISI][Medline]
[Order article via Infotrieve]
-
Wong H, Anderson WD, Cheng T, Riabowol KT. Monitoring mRNA expression by polymerase chain reaction: the "primer dropping" method. Anal Biochem 1994;223:251-258.
[ISI][Medline]
[Order article via Infotrieve]
-
Becker-André M, Hahlbrock K. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res 1989;17:9437-9446.
[Abstract/Free Full Text]
-
Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A 1990;87:2725-2729.
[Abstract/Free Full Text]
-
Mularoni A, Adessi GL, Arbez-Gindre F, Agnani G, Nicollier M. Competitive RT-PCR to quantify CFTR mRNA in human endometrium. Clin Chem 1996;42:1765-1769.
[Abstract/Free Full Text]
-
Apostolakos MJ, Schuermann WHT, Frampton MW, Utell MJ, Willey JC. Measurement of gene expression by multiplex competitive polymerase chain reaction. Anal Biochem 1993;213:277-284.
[ISI][Medline]
[Order article via Infotrieve]
-
Dostal DE, Rothblum KN, Baker KM. An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensin mRNA levels in various tissues. Anal Biochem 1994;223:239-250.
[ISI][Medline]
[Order article via Infotrieve]
-
de Kant E, Rochlitz CF, Herrmann R. Gene expression analysis by a competitive and differential PCR with antisense competitors. Biotechniques 1994;17:934-942.
[ISI][Medline]
[Order article via Infotrieve]
-
Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, et al. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 1994;10:38-47.
[Abstract]
-
Celi FS, Zenilmann ME, Shuldiner AR. A rapid and versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Res 1993;21:1047.[Free Full Text]
-
Morrison C, Gannon F. The impact of the PCR plateau phase on quantitative PCR. Biochim Biophys Acta 1994;1219:493-498.
[Medline]
[Order article via Infotrieve]
-
Pannetier C, Delassus S, Darche S, Saucier C, Kourilsky P. Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation. Nucleic Acids Res 1993;21:577-583.
[Abstract/Free Full Text]
-
Chelly J, Montarras D, Pinset C, Berwald-Netter Y, Kaplan JC, Kahn A. Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells. Eur J Biochem 1990;187:691-698.
[ISI][Medline]
[Order article via Infotrieve]
-
McCulloch RK, Choong CS, Hurley DM. An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. PCR Methods Appl 1995;4:219-226.
[ISI][Medline]
[Order article via Infotrieve]
-
Auboeuf D, Vidal H. The use of the reverse transcription-competitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples. Anal Biochem 1997;245:141-148.
[ISI][Medline]
[Order article via Infotrieve]
-
Henrard DR, Mehaffey WF, Allain JP. A sensitive viral capture assay for detection of plasma viremia in HIV-infected individuals. AIDS Res Hum Retrovir 1992;8:47-52.
[ISI][Medline]
[Order article via Infotrieve]
-
Simmonds P, Balfe P, Peutherer JF, Ludlam CA, Bishop JO, Brown AJ. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol 1990;64:864-872.
[Abstract/Free Full Text]
-
Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med 1998;36:255-269.
[ISI][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
K. A. Warner, E. L. Crawford, A. Zaher, R. J. Coombs, H. Elsamaloty, S. L. Roshong-Denk, I. Sharief, G. V. Amurao, Y. Yoon, A. Y. Al-Astal, et al.
The c-myc x E2F-1/p21 Interactive Gene Expression Index Augments Cytomorphologic Diagnosis of Lung Cancer in Fine-Needle Aspirate Specimens
J. Mol. Diagn.,
August 1, 2003;
5(3):
176 - 183.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
