• Clinical and Experimental Reproductive Medicine
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• 제34권4호
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• pp.275-283
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• 2007

목 적: 본 연구에서는 생리혈에 존재하는 탈락된 자궁내막조직에서의 자궁내막증 관련 유전자들의 발현 양상과 자궁내막증 병태생리와의 관련성을 살펴보고자 하였다. 연구방법: 자궁내막증으로 확진된 환자 (n=16)와 정상 대조군 (n=26)에서 생리주기 2$\sim$3일째 Wallace catheter로 채취한 생리혈로부터 탈락된 자궁내막조직을 분리하였다. 기존의 연구들에서 보고된 12종류의 자궁내막증 관련 유전자들의 mRNA 발현 양상을 semi-quantitative RT-PCR 방법으로 비교, 분석하였다. 결 과: 생리혈에서 분리한 탈락된 자궁내막조직은 조직학적 관찰을 통해 자궁내막조직임을 확인하였다. 총 12가지 종류의 자궁내막증 관련 유전자에 대한 RT-PCR 분석에서 telomerase, c-kit, aromatase등의 mRNA 발현이 관찰되지 않았다. 세포사멸 (apoptosis)과 관련성이 있는 fas, fas ligand, bcl-2, bax 유전자와 stem cell factor, ER-$\alpha$/$\beta$, endometriosis protein-I, secretory leukocyte protease inhibitor 등의 mRNA 발현 양상은 자궁내막증으로 확진된 환자군과 대조군에서 통계적으로 유의한 차이를 나타내지 않았다. 결 론: 결론적으로 자궁내막증과 관련된 다양한 유전자들의 발현 양상을 생리혈에 존재하는 탈락된 자궁내막조직에서 분석하였지만 의미성이 있는 유전자를 동정하지는 못하였다. 이는 자궁내막조직의 생리학적 특징인 생리주기에 따른 유전자 발현의 역동적인 변화와 관련이 있을 것으로 생각된다.

• Toxicological Research
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• 제18권1호
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• pp.59-64
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• 2002

Substantial evidences have been accumulated about the hormone-like effects of exogenous substances such as pesticides and industrial chemicals during past years. The effects of these substances on the endocrine system are believed to be either enhancing or reducing of various endocrine action. It is necessary to identify putative causal agents by the batter system and to assess their ability to disrupt the endocrine system. A variety of in vitro and In vivo approaches have been used to determine the androgenic effects of environmental chemicals. To establish the method for assessment of the putative endocrine disruptors with androgenic activity, we carried out the cell proliferation assay by MTS method after treatment with the various concentration of testosterone in LNCaP cells (human prostatic cancer cell line) and also observed the expression of androgen-related genes by quantitative RT-PCR. In the cell proliferation assay, the results showed that the grouth of LNCaP cells increased within level of at least 10pM testosterone. We measured by quantitative RT-PCR method on the effects of testosterone on mRNA expression of androgen receptor (AR), prostate-specific antigen (PSA), bone morphogenetic protein (BMP) and BMP receptor (BMPR) In LNCaP cells. The results demonstrated that mRNA expression of PSA and BMPR-IB was observed differently within level of at least 0.01 pM testosterone compared with non-treated control. These observations suggest that the detection of PSA and BMPR-IB mRNA by the quantitative RT-PCR in LNCaP cells is very sensitive method to identify the endocrine disruptors to have the androgenic effects.

• 대한소아치과학회지
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• 제42권4호
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• pp.312-318
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• 2015

본 연구의 목적은 치과분야에서 줄기세포 공급원으로써 과잉치의 활용 가능성을 알아보고자 Real Time Quantitative Reverse Transcription Polymerase Chain Reaction (Real Time qRT-PCR)법을 이용하여 발치된 과잉치 치수 유래 줄기세포 (supernumerary dental pulp stem cells, sDPSCs)로부터 상아모세포로의 분화여부를 관찰해 보는 것이었다. 이를 위해 상아모세포의 대표적인 발현인자로 알려진 alkaline phosphatase (ALP), osteocalcin (OC), osteonectin (ON), dentin matrix acidic phosphoprotein 1 (DMP-1) 그리고 dentin sialophosphoprotein(DSPP)의 발현을 분화제 처리 후 0일, 8일 그리고 14일째에 각각 Real Time qRT-PCR 법을 통해 상대적인 mRNA의 발현 양을 비교하여 변화 양상을 알아보았다. 또한 Alizarin-red solution 의 염색을 통해 sDPSCs가 분화제 처리 7일, 14일, 21일 그리고 28일째에 석회화 결절을 형성하는 정도를 시각적으로 확인해 보았다. Real Time qRT-PCR 결과 분화제 처리 8일째에 가장 높은 발현 양을 보이다가 14일째에 감소하는 추세를 나타내었으며, Alizarin-red solution 염색 결과는 7일째부터 흐리게 나타나다가 14일째에는 배지 전반에 걸쳐 진하게 염색되는 소견을 보였다. 따라서, sDPSCs를 이용한 연구에서 Real Time qRT-PCR법을 위한 분화제의 처리 기간은 8일 정도가 적절하며, Alizarin-red solution 염색은 14일이 적당한 것으로 사료된다.

• Genomics & Informatics
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• 제21권4호
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• pp.52.1-52.10
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• 2023

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1280-1286
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• 2011

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at $3.6{\times}10^6$ CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above $10^5$/ml for approx. 10 days, after which cell numbers decreased to levels of $10^3$ CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. $1{\times}10^2$ CFU/ml was detected. The minimum detection level for quantitative PCR in this study was $1{\times}10^2$ to $1{\times}10^3$ CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7621-7628
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• 2013

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.

• Journal of Gastric Cancer
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• 제7권2호
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• pp.59-66
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• 2007

목적: 위암 세포주 및 조직에서 다중 표지자 mRNA 발현을 정량적 RT-PCR 검사를 통해 확인함으로써 이들 표지자를 이용하여 위암의 복강내 미세전이 진단이 가능한가를 평가하고자 본 연구를 시행하였다. 대상 및 방법: 12개의 인체 위암 세포주와 10개의 위암 조직을 대상으로 Carcinoembryonic antigen (CEA), Cytokeratin 20 (CK20), Dopa decarboxylase (DDC), L-3-phosphoserine phosphatase (L3PP)의 네 가지 mRNA를 이용한 정량적 RT-PCR 다중 표지자 분석을 시행하였다. 결과: 12개의 인체 위암 세포주 중 CEA는 4개(33%), CK-20는 1개(8%), DDC는 6개(50%), L3PP는 12개 세포주 모두(100%)에서 과발현되었다. 10개의 위암 조직 중 CEA는 9개, CK20은 3개, DDC는 9개, L3PP는 10개 조직 모두에서 과발현되었다. L3PP는 모든 위암 세포주와 조직에서 과발현을 나타내었으나 과발현 정도는 비교적 낮게 측정된 반면, CEA와 DDC는 일부 위암 세포주 및 조직에서만 과발현을 나타내었지만 파발현 시 충분한 발현도를 나타내었다. 결론: 위암 환자에서 하나 이상의 암 특이적 유전자를 이용한 다중 표지자 분석은 단일 표지자 분석이 가지는 단점을 보완할 수 있을 것으로 예상되며, CEA, DDC, 및 L3PP의 세 가지 mRNA가 후보 유전자로 사용될 수 있을 것으로 생각한다.

• 한국작물학회지
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• 제65권4호
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• pp.496-507
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• 2020

본 연구는 사과 바이러스 ASGV, ASPV 및 ApMV를 각각 정밀하게 진단하고자 SYBR Green I 및 TaqMan probe, 두 종류의 다른 chemical dye를 사용하여 quantitative real-time PCR 검정법을 개발하고자 하였다. 1. 사과 바이러스 ASGV, ASPV 및 ApMV의 국내분리주를 바탕으로 하여 cloning 및 in vitro transcription을 수행해 10배 희석단위 표준시료를 제작하였다. 각 바이러스에 대한 SYBR Green I용 프라이머와 TaqMan probe용 프라이머 및 프로브를 디자인하였다. 2. 상기 제작된 프라이머와 프로브를 이용해 표준시료를 대상으로 real-time PCR을 수행하여 각 바이러스의 증폭곡선과 검량선을 구할 수 있었다. Real-time PCR 결과, SYBR Green I기반의 검정법은 TaqMan probe기반의 검정법 못지 않은 결과를 보여주었으며, 적은 비용에 대량 검정이 요구되는 곳에 효과적으로 응용될 수 있을 것이다. 3. 현장평가를 본 실험에서 개발된 TaqMan probe기반의 real-time PCR검정법과 기존의 RT-PCR검정법과 비교분석하였다. 그 결과 real-time PCR 검정법은 singleplex 및 multiplex RT-PCR보다 더 민감하고 정확한 결과를 내어 RT-PCR로 검출할 수 없는 농도까지 검정할 수 있음을 입증하였다. 4. 본 실험에서 개발한 real-time PCR검정법은 검역현장과 같은 대량의 검사가 요구되는 곳에서는 SYBR Green I 기반의 검정법을, 바이러스 연구분야에서는 세밀한 정량이 가능한 TaqMan probe 방식의 검정법이 활용될 것으로 기대한다.

• Fisheries and Aquatic Sciences
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• 제19권4호
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• pp.21.1-21.11
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• 2016

Background: The evaluation of suitable reference genes as normalization controls is a prerequisite requirement for launching quantitative reverse transcription-PCR (RT-qPCR)-based expression study. In order to select the stable reference genes in abalone Haliotis discus hannai tissues (gill and hepatopancreas) under heavy metal exposure conditions (Cu, Zn, and Cd), 12 potential candidate housekeeping genes were subjected to expression stability based on the comprehensive ranking while integrating four different statistical algorithms (geNorm, NormFinder, BestKeeper, and ${\Delta}CT$ method). Results: Expression stability in the gill subset was determined as RPL7 > RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. On the other hand, the ranking in the subset for hepatopancreas was RPL7 > RPL3 > RPL8 > ACTB > RPL4 > EF1A > RPL5 > RPL7A > B-TU > UBE2 > PPIB > GAPDH. The pairwise variation assessed by the geNorm program indicates that two reference genes could be sufficient for accurate normalization in both gill and hepatopancreas subsets. Overall, both gill and hepatopancreas subsets recommended ribosomal protein genes (particularly RPL7) as stable references, whereas traditional housekeepers such as ${\beta}-tubulin$ (B-TU) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were ranked as unstable genes. The validation of reference gene selection was confirmed with the quantitative assay of MT transcripts. Conclusions: The present analysis showed the importance of validating reference genes with multiple algorithmic approaches to select genes that are truly stable. Our results indicate that expression stability of a given reference gene could not always have consensus across tissue types. The data from this study could be a good guide for the future design of RT-qPCR studies with respect to metal regulation/detoxification and other related physiologies in this abalone species.

• Genomics & Informatics
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• 제21권1호
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• pp.13.1-13.8
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• 2023

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria 'Porphyromonas gingivalis', and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)-based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis-specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.