New primers for methylation-specific polymerase chain reaction enhance
specificity of detecting STAT1 methylation
Ming-Cheng Changa, Ying-Cheng Chiangb, Chih-Ming Hoc, Yu-Li Chenc, Chi-An Chena,
Wen-Fang Chenga,d,e,*, Cheng-Yang Chouf
aDepartment of Obstetrics and Gynecology, Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
bDepartment of Obstetrics and Gynecology, National Taiwan University Hospital Yun-Lin Branch, Douliou City, Yunlin County, Taiwan
cGynecologic Cancer Center, Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan
dGraduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan
eGraduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
fDepartment of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Accepted 2 November 2011
Objective: Signal transducer and activator of transcription (STAT)1 is a key tumor suppressor, which is always methylated in a variety of human
cancers. However, nonspecific primers for the detection of specific promoter hypermethylation of STAT1 gene can lead to false-positive or false-
negative results for gene methylation.
Materials and Methods: We designed new primers for the detection of STAT1 methylation and compared the sensitivities and specificities of
these new primers with prior published primers by methylation-specific polymerase chain reaction (PCR) from ovarian clear cell carcinomas.
The mRNA expression levels of STAT1 in these cancerous tissues were also evaluated by reverse-transcriptase PCR and correlated with the
results of promoter methylation of STAT1 gene.
Results: Nine (39%) of the 23 samples detected by the new primers and 13 samples (56%) detected by prior published primers showed STAT1
methylation. A direct DNA sequencing test revealed that four of the 13 samples (30.8%) showed false positivity for STAT1 methylation using the
prior published primers. In contrast, none of the nine samples was false-positive for the detection of STAT1 methylation using the new primers.
The new primers for the detection of STAT1 methylation showed 100% specificity and 100% sensitivity without false positivity.
Conclusion: Specific primers for methylation-specific PCR are mandatory for the accurate detection of STAT1 gene methylation. Besides,
specific primers can generate correct interpretation of STAT1 gene methylation, and its correlation with the clinicopathological characteristics
and outcome of cancer patients.
Copyright ? 2012, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: methylation; polymerase chain reaction; signal transducer and activator of transcription 1
Hypermethylation in the promoter region of genomic DNA
is now believed to be an important epigenetic alteration
occurring in the early stage of carcinogenesis of many cancers
[1e3]. Cytosineeguanine (CpG) island methylation is one of
the best-understood epigenetic changes in human cancers, and
it is also an important epigenetic mechanism in gene tran-
scriptional regulation . Aberrant methylation of tumor
suppressor genes plays an important role in carcinogenesis, as
a result of altering the DNA secondary structure and inducing
chromosome remodeling, even transcriptional repression [5,6].
Although the methylation status of the CpG island in tumor
* Corresponding author. Department of Obstetrics and Gynecology, National
Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan.
E-mail address: firstname.lastname@example.org (W.-F. Cheng).
Available online at www.sciencedirect.com
Taiwanese Journal of Obstetrics & Gynecology 51 (2012) 43e49
1028-4559/$ - see front matter Copyright ? 2012, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
suppressor genes has been studied extensively for its potential
to predict cancer behavior , this epigenetic alternation has
not proven to be an entirely reliable prognostic marker for
cancer prognosis, due to the exact methods used for detecting
methylation in the CpG island.
Most assays for gene methylation changes rely on the
treatment of genomic DNAwith sodium bisulfite, which could
convert unmethylated but not methylated cytosine residues to
uracil, resulting in the generation of noncomplementary,
single-stranded DNA. Following this conversion process, we
used methylation-specific polymerase chain reaction (MS-
PCR) with a pair of specific PCR primers to detect the
epigenetic alternation of maternal and paternal alleles. MS-
PCR is a frequently used, inexpensive method for detecting
gene methylation. Following bisulfite modification, we per-
formed PCR using two sets of primers designed to amplify
either methylated or unmethylated alleles.
The design of the primers is the crucial issue in obtaining
reliable MS-PCR results for the detection of gene hyper-
methylation. The methylated and ummethylated primer sets
need to be designed for the same CpG sites and include
multiple CpG sites at the 30ends. For this study, we designed
a set of new primers for the MS-PCR to detect signal trans-
ducer and activator of transcription (STAT)1 methylation. The
mRNA expression levels of STAT1 in the ovarian samples
with or without STAT1 gene hypermethylation were also
checked and correlated with the results of the MS-PCR. We
further compared the sensitivity, specificity, and false-positive
and false-negative rates in the detection of STAT1 methyla-
tion by using new and prior published primers. The new
primers that we designed were more accurate in detecting the
STAT1 methylation in ovarian cancer than the prior published
Materials and methods
Patients and specimens
The study was approved by the Institutional Review Board
of the National Taiwan University Hospital. All women were
informed and gave their written consent to participate in the
study. Twenty-three women with three benign ovarian tumors
and 20 with ovarian clear cell carcinomas were enrolled.
Ovarian specimens were obtained intraoperatively and frozen
immediately at e70?C until analysis.
The genomic DNA of the ovarian tissues was isolated using
the Qiagen EZ1 DNATissue Kit (Qiagen, Valencia, CA, USA)
following the manufacturer’s instructions, as described previ-
The MS-PCR was performed as described previously with
some modifications . Briefly, genomic DNA was first
treated with sodium bisulfite, then desulforated with NaOH,
precipitated with ethanol, and resuspended in distilled water.
After the treatment with sodium bisulfate, the methylation
status of the DNA was checked by primers specific for the
methylated and unmethylated alleles of the STAT1 gene. The
processes of MS-PCR in the isolated genomic DNA of ovarian
samples were performed using the EZ DNA Methylation Kit?
(Zymo Research Corporation, Orange, CA, USA), following
the manufacturer’s instructions, as described previously
set of primers were originally designed and reported by Xi 
and have been defined here as the prior published primers.
The primers for detecting unmethylated STAT1 gene were
CAATTAAACACAACTATTCCATA-30(antisense) to generate
a 269-bp product. To detect the methylated STAT1 gene, 50-AA
ATTTGTTTTTTGTTTGGATTTTC-30(sense) and 50-AATT
generate a 266-bp product .
The second set of STAT1 methylated or unmethylated
specific primers that we designed were defined as the new
primers. These primers were designed from the web site http://
sequence published on the Pubmed (accession number:
and 50-AACCAAACAAAAACAACCCAACA-30(antisense) to
generate a 116-bp product. The primers for the methylated
STAT1 gene were 50-TATTTTCGGTATTCGGAATTTTAC-30
(sense) and 50-GAACAAAAACGACCCAACGC-30(antisense)
to generate a 113-bp product.
As shown in Fig. 1, the detection of methylation status of
the STAT1 gene was on the CpG island located at nucleotides
626e895 (prior published primers) and at nucleotides
121e233 (new primers) upstream of the start site of the exon.
The prior primers designed by Xi could detect methylation at
nucleotides 626e895 upstream of the start site of the exon.
(antisense) were used to
Fig. 1. A diagrammatic representation of the MS-PCR specific primers for the
detection of STAT1 promoter methylation. Two sets of methylation-specific
primers for the STAT1 promoter region were used. The prior published
primers could detect the methylation from promoter e895 to e626 with 266-
bp methylated and 269-bp unmethylated PCR products. The new primers
could detect the methylation from promoter e233 to e121 with 113-bp
methylated and 116-bp unmethylated PCR products. The number ranges
above indicate the nucleotide position relative to the translational start site
(ATG). Short vertical bars on the bottom line show the CpG sites.
CpG ¼ cytosineeguanine; MS-PCR ¼ methylation-specific polymerase chain
reaction; STAT1 ¼ signal transducer and activator of transcription 1.
44 M.-C. Chang et al. / Taiwanese Journal of Obstetrics & Gynecology 51 (2012) 43e49
The 266-bp and 269-bp PCR products indicated methylation
and nonmethylation of the STAT1 gene by using the prior
published primers. Our new primers could detect methylation
at nucleotides 121e233 upstream of the start site of the exon.
The 113-bp PCR product indicated methylation of the STAT1
gene. In contrast, the 116-bp PCR product indicated non-
methylation of the STAT1 gene.
The PCR products, whether from the prior published or the
new primers, were further separated by capillary electropho-
resis (CE) or 3% agarose gel electrophoresis (AGE), with the
results being visible after staining with ethidium bromide
(EtBr). We performed direct DNA sequencing to check the
accuracy of the CE and AGE.
CE and direct DNA sequencing
We used CE to analyze the MS-PCR products via the HDA
System with a GCK-5000 cartridge kit (eGene, Irvine, CA,
USA), as described previously . Briefly, the gel-matrix in
the gel cartridge was composed of proprietary linear polymer
with EtBr dye. The PCR products were diluted 20-fold with
deionized water and placed in the sample chamber of the
instrument. DNA samples were then injected into the capillary
channels and subjected to electrophoresis based on the manu-
facturer’s protocol [15e17]. BioCalculator Graphing software
(eGene) was used to label the peak sizes automatically .
The PCR products for CE were also checked by direct DNA
sequence analysis using an automated ABI sequencing system
(ABI3730, Applied Biosystems, Foster city, CA) to confirm
the results of the CE.
Ovarian tissue specimens were collected, frozen, and stored
as described earlier. The total RNA of the ovarian tissues was
isolated using TRIzol reagent (Invitrogen, Carlsbad, CA,
USA) following the manufacturer’s instructions, as described
RNA was first reverse-transcribed to cDNA using the
Moloney murine leukemia virus reverse transcriptase kit
(Invitrogen Life Technologies, San Diego, CA, USA) [18,19].
A set of primers, 50-TTCAGAGCTCGTTTGTGGTG-30and
50-AGAGGTCGTCTCGAGGTCAA-30, were used for 30
cycles to determine the expression levels of STAT1. Glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) was used as
the housekeeping gene to compare with the target gene STAT1.
A set of primers, 50-ACCCAGAAGACTGTGGATGG-30and
50-TGCTGTAGCCAAATTCGTTG30was also used for 30
cycles to generate GAPDH. The PCR products were then
analyzed in 1% agarose gel with EtBr staining in Tris/borate/
EDTA solution. Differences in the transcription levels of
STAT1 between the samples with methylated and unmethy-
lated STAT1 gene were compared using the electrophoresis
Prior published primers for MS-PCR showed a 30%
false-positive rate in the detection of STAT1 gene
We evaluated whether methylation could be observed
within the CpG island of the promoter of the STAT1 gene.
Representative figures of the STAT1 gene methylation by MS-
PCR when using the prior published primers in CE analysis
(Fig. 2A) and in AGE (Fig. 2B) are shown. Thirteen (56.5%)
of the 23 samples showed methylation in the cancerous tissues
and 0/3 in the normal tissues, and the results were consistent
between the CE and AGE analyses.
Direct DNA sequencing was also performed to confirm the
methylation status of the STAT1 using MS-PCR with the prior
published primers. Fig. 2C1, sample S3, revealed unmethyla-
tion of the STAT1 gene methylation by direct DNA
sequencing, but it showed STAT1 gene by MS-PCR using the
prior published primers when analyzed by CE and AGE. In
addition, there were another three samples of unmethylated
STAT1 gene that revealed STAT1 gene methylation by MS-
PCR with prior methylation. A total of four out of 13
samples (31%) showed false-positive results for STAT1 gene
methylation as detected by MS-PCR using the prior published
primers. None of the samples showed false-negative results by
MS-PCR using the prior published primers when checked by
direct DNA sequencing.
New primers for MS-PCR showed no false-positive or
false-negative results for detection of STAT1 gene
We used our new primers to detect methylation of the
STAT1 gene. The primers that we designed are shown in
Fig. 1. The representative figures of MS-PCR using the new
primers by CE and AGE analyses are shown in Figs. 3A and
3B. Nine of the 20 samples (45%) showed methylation in
cancerous tissues. No methylation of the STAT1 gene was
detected in the three normal ovarian tissues. The results were
also consistent between the CE and AGE analyses.
Direct DNA sequencing was performed to confirm the
methylation status of STAT1 using MS-PCR with the new
primers. Sample S3, which revealed nonmethylation of the
STAT1 gene as detected by MS-PCR and CE and AGE anal-
yses, can be seen in Fig. 2B. Direct DNA sequencing also
revealed an unmethylated STAT1 gene, as shown by MS-PCR
with CE and AGE analyses (Fig. 3C2). In addition, the other
three samples that were false-positive for STAT1 gene meth-
ylation using the prior published primers showed non-
methylation of the STAT1 gene using the new primers, and
were confirmed to be unmethylated STAT1 gene by direct
DNA sequencing. When we used our new primers, none of the
23 samples showed false-positive or false-negative results in
the methylation status of the STAT1 gene by MS-PCR. Our
results indicate that the new primers provide more accuracy in
detecting the methylation of the STAT1 gene.
45M.-C. Chang et al. / Taiwanese Journal of Obstetrics & Gynecology 51 (2012) 43e49
RNA expression levels of STAT1 in ovarian cancerous
and normal tissues as detected by RT-PCR
To investigate further the correlation between RNA
expression and methylation status of the STAT1 gene, RT-PCR
was performed. Fig. 4 shows the representative figures of the
RNA expression levels of STAT1 in the tissue samples. The
RNA expression levels of STAT1 (Samples 1 and 2) were
lower in the methylated STAT1 samples than in Samples 3e5,
the unmethylated STAT1 samples. Our results indicated that
the samples with STAT1 gene methylation showed low mRNA
expression of STAT1.
MS-PCR is a PCR-based method for identifying known
allelic mutations in nucleic acid sequences. MS-PCR is
a simple, sensitive and specific method for determining the
methylation status of CpG-rich regions [20,21]. This method is
based on the introduction of artificial mutations into the PCR-
primer binding regions of amplified DNA in an allele-specific
manner. By using allele-specific primers with mutagenic
positions, the artificially introduced mutations can anneal to
the target alleles more specifically. All of the nine STAT1
methylation ovarian cancerous samples could be detected by
MS-PCR in the present study, whether we used the prior
published or the new primers. The MS-PCR products are
specific for their respective alleles and can be directly iden-
tified by AGE or CE without further manipulation [22e24].
Nevertheless, methylation could not be detected by the MS-
PCR using methylation-specific primers when the regions of
the CpG sites were not methylated.
MS-PCR is currently widely used to study the promoter
methylation detection by MS-PCR amplification can produce
false-positive results as we have shown here. In the current
study, four samples without STAT1 gene methylation showed
STAT1 methylation with MS-PCR when using the prior pub-
lished primers. The false-positive results can be attributed to
several reasons. The annealing temperature is the first
important step in an MS-PCR reaction. If the annealing
temperature is too low or too many cycles are used, amplifi-
cation can occur across the 30mismatch . Our new primers
for detecting STAT1 methylation could efficiently amplify the
methylated alleles (Fig. 3). The new primers contained three
CpG sites with the designed annealing temperature at 57?C
during PCR, so the new primers could reduce the wrong PCR
products. In contrast, the prior published primers contained
Fig. 2. STAT1 promoter methylation detected by the prior published primers in various ovarian cancerous samples. (A) CE analysis of PCR products with the prior
published primers. A1: methylated PCR products as indicated by the arrows. A2: Unmethylated PCR products as indicated by the arrows. (B) AGE analysis of the
PCR products using the prior published primers. The S1, S2 and S3 samples showed STAT1 methylation, but the S4 and S5 samples showed STAT1 non-
methylation. (C) Direct DNA sequencing analysis of the STAT1 gene. C1: S3 sample. C2: S4 sample. Both the S3 and S4 samples showed nonmethylation. The
results were incompatible between the CE, AGE and direct DNA sequencing analyses using the prior published primers. AGE ¼ agarose gel electrophoresis;
CE ¼ capillary electrophoresis; PCR ¼ polymerase chain reaction; STAT1 ¼ signal transducer and activator of transcription 1.
46 M.-C. Chang et al. / Taiwanese Journal of Obstetrics & Gynecology 51 (2012) 43e49
only one CpG site with a relatively low annealing temperature
at 54?C, so the PCR products of the prior published primers
could only have comparable specificity. Raising the annealing
temperature resulted in a significantly more efficient amplifi-
cation of the methylated template. By adjusting the annealing
temperature of the PCR amplification, we were able to
improve the specificity of the STAT1 methylation samples.
Another critical parameter affecting the specificity of
methylation-specific PCR is the design of the primers. Suitable
and specific primers can enhance the success rate of the entire
PCR. In general, primers should be designed to amplify
a region that is 80e250 bp in length, and should contain
enough cytosines in the original sequence to assure that
unmodified DNA will not serve as a template for the primers
. The new primers that we designed contained three CpG
sites to amplify a 113-bp DNA fragment. In contrast, the prior
published primers contained only one CpG site to amplify
266-bp nucleotides. Low characterization efficacy in the
primer could be the key issue leading to false-positive results.
By increasing the CpG sites in the primer design, we
approached the appropriate DNA length and were able to
improve the specificity of our assay for STAT1 methylation.
DNA hypermethylation of gene-associated CpG islands
results in either downregulation or complete abrogation of
gene expression. Gene expression should be checked to
confirm the biological effect of gene methylation. Suzuki et al
have reported that the methylation of promoters correlates
with low or no transcription . Many examples also have
demonstrated that methylation of tumor suppressor genes such
as BRCA-1 , RASSF1A , PTEN , and P16 
can result in gene silencing and then carcinogenesis. In the
current study, we also used RT-PCR to confirm the mRNA
expression level and to validate the post-transcriptional
Fig. 4. Expression of STAT1 mRNA in various ovarian cancerous samples. The
expressions of STAT1 mRNAwere lower in the S1 and S2 samples than in the
S3, S4 and S5 samples. STAT1 ¼ signal transducer and activator of tran-
Fig. 3. STAT1 promoter methylation detected by the new primers in various ovarian cancerous samples. (A) CE analysis of the PCR products using the new
primers. A1: methylated PCR products as indicated by the arrows. A2: Unmethylated PCR products as indicated by the arrows. (B) AGE analysis of the PCR
products using the prior published primers. The S1 and S2 samples showed STAT1 methylation, but the S3, S4 and S5 samples showed STAT1 nonmethylation. (C)
Direct DNA sequencing analysis of STAT1 gene. C1: S1 sample. C2: S3 sample. S1 was STAT1 methylation and S3 was STAT1 unmethylation. The results were
compatible between the CE, AGE and direct DNA sequencing analyses using the new primers. AGE ¼ agarose gel electrophoresis; CE ¼ capillary electrophoresis;
PCR ¼ polymerase chain reaction; STAT1 ¼ signal transducer and activator of transcription 1.
47 M.-C. Chang et al. / Taiwanese Journal of Obstetrics & Gynecology 51 (2012) 43e49
modification of STAT1. The expression levels of STAT1
mRNA in these unmethylated STAT1 samples remained high
(Fig. 4). In contrast, the samples with methylated STAT1
demonstrated downregulated expression of STAT1 mRNA
(Fig. 4). Consequently, the STAT1 methylation has the bio-
logical function of reducing STAT1 gene expression.
Analysis of the hypermethylation of gene promoters has
extraordinary importance for tumor biology and clinical
applications. Methylated promoters have been detected in
a variety of cancer patient tissues, as well as in ovarian cancer
tissues [1,31e33]. Studies have demonstrated that profiles of
specific promoter methylation in different tumors are associ-
ated with the clinical response and outcomes of cancer patients
[2,34,35]. However, these methylation-related biomarkers are
still limited in their clinical application, because of the lack of
specificity and sensitivity. This highlights the critical need for
the design of specific primers and probes to improve the
sensitivity and specificity for the detection of gene methyla-
tion. With the identification of DNA methylation in cancer
initiation and progression, distinct efforts should be made to
develop strategies for facilitating clinical application.
AGE and CE are the common analytic methodologies for
the analysis of PCR products. We utilized CE to demonstrate
our MS-PCR results in this study. AGE is most commonly
used for the separation of biological macromolecules and is
a visual method for confirming the presence of nucleotides. It
provides good resolution and separation of large molecules
from small. However, the visualization of oligonucleotides
requires the highly genotoxic agent EtBr. This test is time-
consuming, requires a larger sample volume, and its high
pollution level should be noted. CE showed technical advan-
tages over AGE and previous gel methods in terms of the time
and labor saved by the automated instrumentation . The
most important advantage is its ability to measure the size of
PCR products with very high resolution . Our data from
the MS-PCR and CE revealed the same sensitivity and spec-
ificity with those from direct genomic DNA sequencing
(Fig. 3). The major disadvantages of CE including non-
covalent complexes are frequently disrupted results  and
its expensive instrumentation. However, if considered as high-
throughput equipment for further clinical application, CE is
a powerful and attractive strategy for methylation analysis.
In the present study, new primers of MS-PCR could detect
the methylation of STAT1 gene more accurately than the prior
published primers. The mRNA expression levels of STAT1
gene detected by RT-PCR also confirmed the accuracy of the
new primers of MS-PCR. The designation of specific primers
is indeed an important issue for the accurate detection of
STAT1 gene methylation. Correct detection of the methylation
of tumor suppressor gene STAT1 can be efficiently utilized for
the correlation of clinicopathological characteristics and for
the prediction of the outcome of ovarian cancer patients.
This work was supported by a grant from National Taiwan
University Hospital (NTUH100-S1609). We would like to
thank the Second and Seventh Core Laboratories of the
Department of Medical Research at National Taiwan Univer-
sity Hospital for technical assistance.
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