RAPID COMMUNICATION
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Progress toward complete sequencing of all human genes through the Human Genome Project has already
resulted in a need for methods that allow quantitative expression measurement of multiple genes simultaneously. It is increasingly recognized that relative measurement of multiple genes will provide more mechanistic information regarding cell pathophysiology than measurement of individual genes one by one or by
methods that do not allow direct intergene comparison. In this study, previously described quantitative reverse transcription-polymerase chain reaction methods were modified in an effort to provide a rapid, simple method for this purpose. Internal standard competitive templates (CTs) were prepared for each gene
and were combined in a single solution containing CTs for more than 40 genes at defined concentrations
relative to one another. Any subsequent dilution of the CT mixture did not alter the relationship of one CT
to another. Because the same CT standard solution or a dilution of it was used in all experiments, data obtained from different experiments were easily compared. The use of multiple CT mixtures with different
housekeeping gene to target gene ratios provided a linear dynamic range spanning the range of expression
of all genes thus far evaluated. CT stock solutions were used to simultaneously quantify the expression of
25 genes relative to 
-actin and glyceraldehyde-3-phosphate dehydrogenase in normal and malignant
bronchial epithelial cells. Because the CT concentrations were known, data in the form of both absolute
messenger RNA (mRNA) copy number and mRNA relative to housekeeping gene mRNA were obtained.
The methods and reagents described will allow rapid, quantitative measurement of multiple genes simultaneously, using inexpensive and widely available equipment. Furthermore, the CT standard solution may be
distributed to other investigators for interlaboratory standardization of experimental conditions.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Several different methods for measurement of multiple
genes simultaneously have been introduced over the last
two years, including SAGE (1) and two forms of high-density oligonucleotide arrays (2, 3). These methods are still
in development, are not widely available, rely on expensive equipment, are relatively insensitive, and at the present
time are labor-intensive. The high-density array technology has potential for large-scale measurement of all human genes simultaneously, but in its current form requires at least 1 µg of RNA for each experiment (2). We have developed a quantitative reverse transcription-polymerase
chain reaction (RT-PCR) method that is not labor-intensive, does not require expensive equipment, and allows the
rapid quantitative measurement of many genes simultaneously, using nanogram amounts of complementary DNA
(cDNA) (4, 5). In an effort to scale up this method for the
simultaneous measurement of large numbers of genes, we have thus far prepared internal standard competitive templates (CTs) for more than 100 genes. Here we describe the
preparation of reagents, evaluation of experimental reproducibility and linear dynamic range, and application of the
methods for simultaneous measurement of 25 genes relative to -actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured human bronchial epithelial
cells (BEC).
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Reagents
PCR buffer (10×) was obtained from Idaho Technology,
Inc. (Idaho Falls, ID). Taq polymerase (5 U/µl), M-MLV
reverse transcriptase, M-MLV RT 5× buffer (250 mM
Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2; 50 mM
dithiothreitol), oligo dT primers, RNasin, pGEM size marker, and deoxynucleotide triphosphates (dNTPs) were
obtained from Promega (Madison, WI). Seakem LE and
Nusieve agarose were obtained from FMC Bioproducts
(Rockland, ME). TRI-REAGENT was obtained from Molecular Research Center, Inc. (Cincinnati, OH). Ribonuclease (RNase)-free water was obtained from GIBCO-BRL
(Grand Island, NY). RPMI-1640 medium was obtained
from Sigma (St. Louis, MO) and bronchial epithelial cell
growth medium (BEGM)
which is bronchial epithelial
cell basal medium containing bovine pituitary extract (52 µg/
ml), insulin (5 µg/ml), hydrocortisone (0.5 µg/ml), GA-1000
(0.1%), retinoic acid (0.1 ng/ml), transferrin (0.1 mg/ml), triiodothyronine (6.5 ng/ml), epinephrine (0.5 µg/ml), and
human epidermal growth factor (0.5 ng/ml)was obtained
from Clonetics (San Diego, CA). LHC basal medium was
obtained from Biofluids (Rockville, MD), and natural human fibronectin and type 1 rat-tail collagen were purchased
from Collaborative Biomedical Products (Bedford, MA).
All other chemicals and reagents were molecular biology
grade.
Cells
Lung carcinoma cell lines NCI-H446, NCI-H82, NCI-N417, A549, A427, NCI-H2126, Calu-1, SW900, and NCI-H520 (American Type Culture Collection, Rockville, MD) were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1 mM glutamine. Normal human bronchial epithelial cells (BEC) (Clonetics) lot numbers 10525 (49-yr-old Caucasian female), 17378 (10-yr-old Caucasian male), 17684 (20-yr-old Caucasian male), 6F0333 (41-yr-old Caucasian female), and 6F0450 (16-yr-old Hispanic female) were obtained from Clontics at passage two and were cultured as described (6) in the presence of serum-free BEGM. To incubate normal cells under the same conditions as tumor cells, BEGM was removed from cultured normal BEC and replaced with RPMI-1640 containing 10% FBS and 1 mM glutamine 16 to 18 h prior to RNA extraction. All cells were incubated at 37°C in the presence of 5% CO2 in 60-mm petri dishes that were coated with a solution of 10 ng/ml natural human fibronectin and 50 ng/ ml type 1 rat-tail collagen in LHC basal medium.
Sample Preparation and RNA Extraction
After cultured cells had reached 70 to 90% confluence,
the medium was removed by aspiration and 1 ml TRI-
REAGENT was added to each dish to lyse the cells, denature the proteins, and release the nucleic acids. Total RNA
extraction and RT were performed as described by the
TRI-REAGENT manufacturer and previously described methods (7). The cDNA samples were balanced with the
CT mixture by dilution with TE buffer (10 mM Tris, 1 mM
ethylenediamenetetraacetic acid) such that 1 µl, when included in a 10-µl PCR reaction containing 5 × 10
14 M
-actin CT and -actin primers, yielded both native and
CT -actin PCR products that were approximately equally
intense on ethidium bromide-stained gels.
Quantitative PCR Amplification
Primers and CT standard mixtures.
Oligo software was
used to select primers based on the absence of stable duplex formation, low likelihood of false priming sites, and
an optimal annealing temperature of 58°C (Table 1). When primers selected on this basis did not provide sufficient amplification (approximately 1 in 6 pairs), a new pair
was selected. Rarely, it was necessary to prepare a third
pair of primers. Amplification efficiency was considered
sufficient when the amount of native template in 1 µl
cDNA that had been diluted as described above (see SAMPLE PREPARATION AND RNA EXTRACTION section) and
as little as 1018 M target gene CT could be co-amplified
and visualized on an ethidium bromide-stained agarose
gel. Primers for all target genes and the two housekeeping
genes that were evaluated in this study are listed in Table
1. Information regarding these as well as all other primers
for CTs (a total of 40) that were included in the CT mixtures described here will be made available on the World Wide Web (http://darwin.mco.edu/medicine; click on Affiliated Groups and then on Pulmonary Research Lab).
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-actin, 191) from
the position of the bp at the 5' end of the CT primer (for
-actin, 587) and adding the length of the reverse primer
(for -actin, 20). Thus, in the case of -actin, the value
would be derived as follows: 587 
191 + 20 = 416 bp.
The concentration of gel-purified CTs (in 30 µl of TE)
was determined by electrophoresis of 1 µg pGEM size
marker next to three lanes, each containing 1 to 5 µl of the
purified CT PCR product through a 4% (3% Nusieve, 1%
Seakem LE) agarose gel containing 0.5 µg/ml ethidium
bromide. Bands were visualized with a Foto/Eclipse (Fotodyne, Hartland, WI) image analysis system, and densitometric analysis of digital images was performed using Collage software (Fotodyne) and a Power Mac 7100/66 (Apple
Computer, Cupertino, CA). CT molarity was calculated
after comparing the pixel quantity for the CT bands with
that of a known amount of one of the pGEM fragments
according to previously described methods (4).
CT mixtures derived from the same original stock were
used for all of the data reported here. All CTs were combined in a mixture that contained 5 × 1012 M -actin and
1011 M of all other CTs, including GAPDH in TE buffer.
This mixture was diluted 100-fold with 5 × 1012 M -actin
in TE buffer to make an A5 × 10e 12/G13/O13 mixture
where A is the CT molarity for -actin, G represents GAPDH, and O is all other genes. A5 × 10e 12/G13/O13
was then diluted 10×, 100×, and 1,000× with A5 × 10e 12
M/1013 M -actin/GAPDH CT mixture in TE buffer to
make A5 × 10e 12/G13/O14, A5 × 10e 12/G13/O15, and
A5 × 10e 12/G13/O16 mixtures, respectively. These solutions were then diluted 10× with TE buffer to give typical
final working solutions that were A5 × 10e 13/G14/O15,
A5 × 10e 13/G14/O16, and A5 × 10e 13/G14/O17. A 1-µl
volume of the desired mixture is included in each 10-µl PCR reaction; thus, the actual CT molarity in each reaction is 10-fold less than that in the working mixture. Because
all dilutions were carried out after all CTs had been combined, the relationship of any target gene CT to any other
target gene CT in all dilutions was the same as in the original mixture containing 5 × 1012 M -actin and 1011 M all
other CTs. Thus, data obtained from experiments that involved the use of different dilutions of this original CT
mixture were directly comparable. It is important to note
that each of the primer pairs for a particular gene amplifies a single band from the CT mix when no native cDNA
is present.
Although most PCR reactions reported here were conducted in an air thermocycler, many of the primer pairs
were also used in an MJ PTC-100 block model (MJ Research, Inc., Watertown, MA) without significant difference in efficiency.
Preparation of PCR reaction mixtures.
Reaction volumes
were 10 µl and each contained 0.05 µg of each primer, 0.5 U Taq polymerase, 1 µl PCR buffer, 0.2 mM dNTPs, water, 1 µl of a CT mixture containing the desired molarity of each CT, and 1 µl cDNA diluted such that native -actin
competed equally with the -actin CT present in the chosen CT mixture. To amplify each gene and its CT in individual reactions, each primer pair was placed in separate
tubes and then mixed with an aliquot of the master mixture containing all other components of the reaction. The
entire volume of mixed reaction mixture was transferred
to a capillary tube and the open ends of the tube were
heat-sealed. The reaction mixtures were cycled in a Rapidcycler air thermocycler as described above and electrophoresed on a 3% Nusieve/1% SeaKem agarose gel, and
quantitative analysis was performed as described below.
Levels of expression were reported as units of messenger
RNA (mRNA)/106 -actin mRNA molecules.
-actin/target gene ratio for an experiment was based on
the relative cDNA concentration in the sample being analyzed and on the relative expression of the target gene
compared with the housekeeping genes. The housekeeping genes used for all data reported in this study were
-actin and GAPDH.
Samples of cDNA were diluted such that 1 µl competed
equally with 105 to 106 molecules of -actin CT or 103 to
104 molecules of GAPDH CT. To prepare a reaction mixture containing 3 × 105 molecules of -actin CT, 1 µl of
CT mixture containing -actin CT at a concentration of
5 × 1013 M was included in a 10-µl reaction, giving a final
concentration of 5 × 1014 M. When cDNA samples were
in balance with 5 × 1014 M -actin CT, the concentration
of target gene CT that was approximately in balance with
the corresponding native template varied from one gene
to another (Figure 1) and from one cell type to another. For the normal BEC population analyzed in Figure 1, the
appropriate CT concentration in the reaction for CDK4
was 1015 M (which in a 10-µl reaction volume amounted
to 6,000 molecules), for p21 and spr1 it was 1016 M (600 molecules in 10 µl), for GADD45 it was 1018 M (6 molecules in 10 µl), and for all other genes it was 1017 M (60 molecules in 10 ml).
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Digital quantitation of PCR products and calculation of gene expression. The initial step in quantitation was digital image analysis. We analyzed pGEM size marker on every gel to identify the range for which the relationship between DNA fragment size (in bp) and ethidium bromide staining intensity (in pixels) was linear. An example of such an analysis is provided in Figure 2. On the basis of this control, we could ensure that the bands being analyzed were all within the dynamic range of the image analysis system. When a band was not within the linear range, we could increase or decrease the amount of cDNA or CT in the reaction mixture and/or change the relationship between housekeeping gene and target gene CTs. The bands were then quantified on the basis of total ethidium bromide staining.
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-actin homodimer pixel quantity was corrected for size as follows: 712,800 pixels × 416 bp
(CT size)/532 bp (native size) = 557,377 pixels.
A third band that migrated between the native and CT
PCR products (Figures 1 and 3) was often observed. This
band represented a heterodimer (HD) that formed between one strand of native and one strand of CT, and its
intensity was related to the amount of PCR product loaded.
The HD, if present, was taken into account when determining mRNA expression. For the example shown in Figure 3, the -actin HD pixel intensity was corrected for size
as described previously (to 119,749 pixels). Next, half of
the HD pixel value was added to the corrected native pixel
value (557,377 pixels + 59,875 pixels = 617,252 pixels) and
half was added to the CT pixel intensity (690,592 pixels + 59,875 pixels = 750,467 pixels). The initial number of native -actin molecules present in the PCR reaction then
was determined by multiplying the ratio of the corrected
native to CT -actin pixel intensities by the initial number
of -actin CT molecules present in the PCR reaction (3 × 105 molecules): 617,252 pixels/750,467 pixels × 3 × 105 molecules = 246,747 molecules.
The initial number of native CDK4 molecules present
in the PCR reaction was determined in the same manner.
The native pixel intensity was first corrected for size:
418,080 pixels × 310 bp/396 bp = 327,285 pixels. The HD
was then corrected for size as described previously (to
29,334 pixels), and half of this value was added to the corrected native pixel value and to the CT pixel value. The ratio of these corrected native and CT band intensities was multiplied by the initial number of CDK4 CT molecules
present in the reaction (6 × 103 molecules): 341,952 pixels/
147,723 pixels × 6 × 103 molecules = 13,889 molecules.
Thus we determined that there were 13,889 molecules of
CDK4 mRNA per 246,747 molecules of -actin mRNA in
this sample, which may be converted to 56,288 molecules of CDK4 mRNA per 106 molecules of -actin mRNA
(Figure 1).
Experiments were performed in duplicate or triplicate
unless otherwise stated (see footnotes for Tables 2 and 3).
Because all experiments were performed with dilutions of
the same CT mixture (i.e., the ratio of any target gene CT
to any other target gene CT was constant in all experiments), and because amplification efficiencies of all native
sequences relative to all corresponding CT sequences
were approximately equal for all genes thus far evaluated (Figure 4 and data not shown), we were able to detect intersample differences in the expression of many individual
genes as well as alterations in the expression of each gene
relative to all other genes.
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Data Analysis
All statistical analyses were carried out using SAS software (version 6.11, 1996; SAS Institute, Cary, NC). The natural log of all expression values was used to normalize all data for statistical comparison. This was necessary because of the wide fluctuations in gene expression among individuals within populations (e.g., c-myc in small-cell carcinoma, E2F-1 in normal, and p21 in small-cell and squamous cell populations).
Cell Size Determination
Cultured cells were assessed digitally using an Olympus IMT2 inverted microscope and Collage 4.0 or NIH image 1.59 software. The average number of pixels per cell was determined from at least 25 cells in each population.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Results from a Typical Experiment
A typical quantitative experiment that provided data for
26 genes including -actin and GAPDH in cultured normal
BEC lot 17378 incubated in BEGM is shown in Figure 1.
For this experiment all genes were quantifiable except for
cyclin D1 and NSE. The cyclin D1 primers that were chosen were inefficient, and thus neither the native nor the
CT amplified sufficiently for quantification. Subsequently,
additional cyclin D1 primers with greater efficiency were
identified. A new cyclin D1 CT that was amplified by the second set of primers was prepared and has been included
in subsequent CT mixtures. NSE was not quantifiable in
this experiment because the CT concentration of 1018 M
was too low to compete with the amount of native template present. NSE was quantified in this sample by including CT molarities of A5 × 10e 14/G15/O17 in subsequent
PCR reactions.
Amplification Efficiency of Native Relative to Varying Amounts of CT
The reliability of this method depends in part on a linear
relationship between the amplification of native sequences
relative to CT sequences. We have examined this relationship for 16 genes including -actin, GAPDH, c-myc, E2F-1,
and p21 (Figure 4 and data not shown). Because all correlation coefficients were close to 1, we were confident in determining gene expression when the imbalance between
native and CT band densities was up to 10-fold. When the
imbalance between native and CT band densities was
greater than 10-fold, we selected a CT mixture with a more
appropriate -actin/target gene ratio for repeat analysis.
Effects of Unexpected PCR Products on Quantitation
For some genes (e.g., p53 and Bax-
) a larger, unexpected
PCR product representing either an alternatively spliced
transcript or a cDNA sequence derived from another gene
that contains sequence homology to the relevant primers
was observed (Figure 1). No bands were visible between
the native and unexpected bands. This indicates that no
HD between the native or CT sequences and the unexpected product was formed, probably because there was not
sufficient sequence homology. It is possible that co-amplification of the alternate sequence will reduce the amount of
native and CT sequence amplified because of competition
for primers, but there is no reason why it would alter the
relative amplification efficiency of native and CT. It was
possible to compare the unexpected amplification product
with CT amplification to determine whether there was any intersample difference in expression. No significant associaton with malignancy was observed.
Reproducibility
Between five and 14 replicate measurements of GAPDH
relative to -actin expression were performed with cDNA
samples from the 19 cell populations represented in Tables
2 and 3. The standard deviation (SD) was 
25% of the
mean in nine samples and 50% of the mean in all but
one sample (in which the SD was 52% of the mean). Thus,
in most experiments, it will be possible to identify 2-fold
changes in gene expression with confidence.
Accuracy
Knowledge of the value of the slope in these experiments is a prerequisite to accurate determination of gene expression and accurate determination of the relative expression of one gene to another. Because the slope has not been higher than 2 or lower than 0.5 in a total of 16 genes evaluated thus far (Figure 4 and data not presented), the effect of differences in the slope is small relative to intersample variation in expression.
GAPDH/-actin Ratio Relationship to Cell Size
There is variation in the GAPDH/-actin ratio from one
cell sample to another (Table 2). Because the ratio tended
to be higher in the malignant cell lines, we hypothesized
that intercell population variation in the ratio was related
to cell size. A significant relationship (P < 0.001) between
two-dimensional cell surface area in culture and GAPDH/
-actin ratio was observed (Figure 5).
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Quantitation of Gene Expression by Multiplex or Nonmultiplex RT-PCR
The CT mixture described here can be used to quantitatively measure gene expression either by multiplex competitive RT-PCR (4, 5) or by nonmultiplex competitive RT-PCR. In multiplex RT-PCR, the native and CTs for both the housekeeping gene and the target gene are amplified in the same reaction. For nonmultiplex RT-PCR, the experiment is designed the same as for multiplex RT-PCR except that primers for only one gene are included in each PCR reaction tube. Because of the constant relationship between CT concentrations in each experiment and the use of master mixtures, the results obtained by either method are highly reproducible. A protocol for quantitative nonmultiplex RT-PCR was developed because multiplex RT-PCR does not always work in air thermocyclers, yet the use of air thermocyclers significantly reduces the amount of time necessary for each experiment. Assessment of a single sample by multiplex or nonmultiplex RT-PCR in an MJ PTC-100 thermal induction thermocycler (MJ Research, Inc.) was compared with nonmultiplex RT-PCR in a Rapidcycler air thermocycler (Idaho Technologies; Figure 6). The number of CYP2A6 mRNA molecules per 106 GAPDH mRNA molecules was determined for each PCR method (see legend to Figure 6), a mean was determined for the results, and the standard deviation was less than 50% of the mean. Therefore, nonmultiplex competitive RT-PCR in an air thermocycler was used to obtain all of the data reported here because it was far less time-consuming and labor-intensive. When the available amount of cDNA is limited, multiplex competitive RT-PCR is preferable because both the housekeeping gene and the target gene can be quantified from the same cDNA aliquot.
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Genomic DNA Contamination
Detecting genomic DNA contamination in RT-PCR reactions was discussed previously (4). Whenever possible, we designed primers for RT-PCR that flank or cross introns in the genomic sequence. Thus, if genomic DNA did contaminate the RNA preparation, and this genomic DNA was carried over through the RT into the cDNA, the PCR product (if amplified) derived from genomic DNA would be larger than the PCR product derived from cDNA. Bands that might represent genomic DNA contamination were not observed in any of the experiments reported here. Circumstances that would increase the chances of significant interference in cDNA quantitation from contaminating genomic DNA include (1) an RNA preparation with a high level of genomic DNA contamination, and (2) a low expression of the gene being assessed.
Thus, although others have advocated including a DNase step following RNA extraction, it is not necessary (and is potentially highly deleterious by increasing the chance of losing sample and contaminating other reagents with DNase) when using the methods described here. However, it is advisable to assess each new cDNA preparation for genomic DNA contamination by including undiluted cDNA in a PCR reaction along with primers for a gene known to be expressed at low levels and that flank an intron. In these experiments, primers for PCNA, MUC1, and GADD45 flanked an intron and each was expressed at low levels in many of the samples evaluated in this study. Amplification of genomic DNA with these primers would produce larger PCR products than amplification of cDNA. No bands were observed that would correspond to genomic DNA following amplification and electrophoresis using any cDNA sample evaluated in this study.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Here is described a quantitative competitive RT-PCR method using standardized mixtures of CTs to measure rapidly and accurately the mRNA expression of many genes simultaneously. This method allows the detection of not only variation in gene expression levels from one sample to another, but also the intergene mRNA stoichiometric relationships in the same sample. It is hypothesized that intergene expression alterations will be more mechanistically related to basic phenotypic differences than variation in expression of individual genes. This hypothesis was tested by assessing the relative expression of 25 genes involved in cell cycle control, apoptosis, DNA repair, and differentiation compared with the expression of two housekeeping genes in nine cultured human lung tumor cell lines versus five cultured normal BEC samples. One important observation was that the gene expression index c-myc × E2F-1/p21 better discriminated between normal and malignant BEC than the expression of any single cell cycle gene (10). Thus, data obtained in those experiments support our hypothesis. Another important observation was that the spr1 gene was significantly expressed and inducible in all cultured normal BEC evaluated, whereas it was neither expressed nor inducible in all carcinoma cell lines evaluated (11).
We are preparing reagents for additional genes and plan to combine them in batches of approximately 50. Theoretically, it would be possible to prepare a mixture containing CTs for all human genes (50-100,000), but for most experiments in which fewer genes will be evaluated the CTs for the genes not being studied would be wasted. CT mixtures for all human genes may be desirable when using the method in combination with high-density oligonucleotide arrays.
It is not yet known how many genes must be measured to define optimally any particular phenotype. This likely will depend on the cell type and specific goals of the investigator. For most purposes, it is possible that measurement of no more than 500 genes will be necessary to define a phenotype optimally. If this is the case, using the methods described here, 500 µl of CT mixture and 500 ng of cDNA would be needed for a single experiment. However, through miniaturization and use of capillary electrophoresis it may be possible to reduce the amount needed to 50 µl and 50 ng, respectively.
The method used in these studies allows rapid, quantitative measurement of many genes simultaneously and it allows direct comparison of gene expression with that of an internal standard. The same standards can be made available to interested laboratories, allowing development of databases with a common reference standard.
The CT mixtures described here and similar CT mixtures have been used to evaluate expression of the genes described in this paper as well as cytokine, antioxidant enzyme, and xenobiotic metabolism genes in primary normal and malignant lung cells, including bronchial brush, alveolar macrophage, lung parenchyma, and primary bronchogenic carcinoma specimens (4 and J. C. Willey, unpublished data).
The reproducibility described here will allow detection of differences in expression from one sample to another of 2-fold or greater. This is equivalent to or better than that observed with most other measures of gene expression, including Northern blotting and RNase protection assays. Based on data from Figure 1, the GAPDH value was reproducible within 30% even when three different CT mixtures were used. Although 30% may seem like a large variation for most assays, it is better than could be expected with Northern analysis. The method described here is also more sensitive than other gene expression methods. For example, in typical experiments 106 cells yield enough RNA (approximately 2 to 5 µg) to provide cDNA for 5 × 102 to 5 × 103 assays. Thus, with the method described here, multiple gene expression studies can be conducted when a limited amount of tissue is available. In contrast, the same amount of RNA typically would not be sufficient for one Northern blot assay or for more than one or two RNase protection assays. Furthermore, with this method, as opposed to Northern blot or RNase protection assays, it is possible to obtain more replicates because each experiment requires 4 h as opposed to as many as 4 d, and the method is far less prone to lab error. With larger numbers of replicates it is expected that the standard deviation would decrease and the ability to identify small differences in gene expression would increase.
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Footnotes |
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Address correspondence to: Dr. James C. Willey, Div. of Pulmonary and Critical Care Medicine, Dept. of Medicine, Medical College of Ohio, 3000 Arlington Ave., Toledo, OH 43699-0008. E-mail: jwilley{at}opus.mco.edu
(Received in original form June 21, 1997 and in revised form January 12, 1998).
* These authors have contributed equally to this manuscript.Acknowledgments: These studies were funded by the following grants: NIEHS R01 05719 and NIEHS P01 01640.
Abbreviations BEC, bronchial epithelial cells; BEGM, bronchial epithelial cell growth medium; bp, base pair(s); CT, competitive template; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HD, heterodimer; RNase, ribonuclease; RT-PCR, reverse transcription-polymerase chain reaction; TE buffer, 10 mM Tris, 1 mM ethylenediamenetetraacetic acid.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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1.
Velculescu, V. E.,
L. Zhang,
B. Vogelstein, and
D. W. Kinzler.
1995.
Serial
analysis of gene expression.
Science
270:
484-487
2. Lockhart, D. J., H. Dong, M. C. Byrne, M. T. Follettie, M. V. Gallo, M. S. Chee, M. Mittmann, C. Wang, M. Kobayashi, H. Horton, and E. L. Brown. 1996. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 14: 1675-1680 . [Medline]
3.
Schena, M.,
D. Shalon,
R. W. Davis, and
P. O. Brown.
1995.
Quantitative
monitoring of gene expression patterns with a complementary DNA microarray.
Science
270:
467-470
4.
Willey, J. C.,
E. L. Coy,
M. W. Frampton,
A. Torres,
M. J. Apostolakos,
G. Hoehn,
W. H. Schuermann,
W. G. Thilly,
D. E. Olson,
J. R. Hammersley,
C. L. Crespi, and
M. J. Utell.
1997.
Quantitative RT-PCR measurement of
cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and
NADPH oxidoreductase expression in lung cells of smokers and nonsmokers.
Am. J. Respir. Cell Mol. Biol.
17:
114-124
5. Apostolakos, M. J., W. H. T. Schuermann, M. W. Frampton, M. J. Utell, and J. C. Willey. 1994. Measurement of gene expression by multiplex competitive polymerase chain reaction. Anal. Biochem. 213: 277-284 .
6. Lechner, J. F., and M. A. LaVeck. 1985. A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J. Tissue Culture Methods 9: 43-48 .
7. Willey, J. C., E. Coy, C. Brolly, M. J. Utell, M. W. Frampton, J. Hammersley, W. G. Thilly, D. Olson, and K. Cairns. 1996. Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells. Am. J. Respir. Cell Mol. Biol. 14: 262-271 [Abstract].
8.
Gilliand, G.,
S. Perrin,
K. Blanchard, and
H. F. Bunn.
1990.
Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction.
Proc. Natl. Acad. Sci. USA
87:
2725-2729
9.
Celi, F. S.,
M. E. Zenilman, and
A. R. Shuldiner.
1993.
A rapid and versatile
method to synthesize internal standards for competitive PCR.
Nucleic Acids Res.
21:
1047
10.
DeMuth, J. P.,
D. A. Weaver,
E. L. Crawford,
C. M. Jackson,
J. C. Hoban,
S. A. Khuder, and
J. C. Willey.
1998.
The gene expression index c-myc × E2F-1/p21 is highly predictive of malignant phenotype in human bronchial
epithelial cells.
Am. J. Respir. Cell Mol. Biol.
19:
18-24
11.
DeMuth, J. P.,
D. A. Weaver,
E. L. Crawford,
C. M. Jackson, and
J. C. Willey.
1998.
Loss of spr1 expression measurable by quantitative RT-PCR in
human bronchogenic carcinoma cell lines.
Am. J. Respir. Cell Mol. Biol.
19:
25-29
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