자궁경부암은 전 세계적으로 네번째를 차지하는 여성암이다. 자궁경부암의 대부분 원인은 인유두종 바이러스의 감염이다. 인유두종 바이러스를 검출하기 위해 다양한 분자진단학적 방법들이 고안되었다. 분자진단학적 방법 중의 real-time PCR은 목표 DNA 또는 RNA의 정량과 민감도 향상을 목표로 도입되었다. 특히, real-time PCR 과정은 수행 전에 RNA 추출 및 상보적인 DNA 합성 과정이 필요하다. 따라서 본 연구에서는 민감하고 적합한 상보적인 DNA 합성 키트를 알아보기 위해서 상보적인 DNA 합성에 이용되는 두 개의 상용화된 키트를 평가하였다. 자궁경부암 세포주에서 두개의 상보적인 DNA 합성 키트의 $R^2$과 효율성을 비교한 결과 차이가 없었다. 그러나 Invitrogen 키트보다 Takara 키트가 $10^2$ 및 $10^3$ SiHa 세포주에서 P<0.001를 나타내었고 $10^1$ 및 $10^2$ HeLa 세포주에서도 P<0.001를 나타내었다. 이를 통해 Takara 키트가 Invitrogen키트보다 민감도가 높음을 알 수 있었다. 또한 40개의 탈락세포검체의 8, 4, 2, 1 mL을 이용하여 상보적인 DNA 합성 키트를 비교한 결과 Invitrogen 키트보다 Takara 키트가 8, 4, 1 mL에서 P<0.01 및 0.5 mL에서 P<0.05을 나타내어 임상 검체를 이용하였을 때에도 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수 있었다. 본 연구는 적합한 상보적인 DNA 합성 키트를 확인하기 위해 수행되었으며, 상보적인 DNA 합성 키트가 real-time PCR 결과 다양성에 영향을 미친다는 것을 시사하였다.
자궁경부암은 전 세계적으로 네번째를 차지하는 여성암이다. 자궁경부암의 대부분 원인은 인유두종 바이러스의 감염이다. 인유두종 바이러스를 검출하기 위해 다양한 분자진단학적 방법들이 고안되었다. 분자진단학적 방법 중의 real-time PCR은 목표 DNA 또는 RNA의 정량과 민감도 향상을 목표로 도입되었다. 특히, real-time PCR 과정은 수행 전에 RNA 추출 및 상보적인 DNA 합성 과정이 필요하다. 따라서 본 연구에서는 민감하고 적합한 상보적인 DNA 합성 키트를 알아보기 위해서 상보적인 DNA 합성에 이용되는 두 개의 상용화된 키트를 평가하였다. 자궁경부암 세포주에서 두개의 상보적인 DNA 합성 키트의 $R^2$과 효율성을 비교한 결과 차이가 없었다. 그러나 Invitrogen 키트보다 Takara 키트가 $10^2$ 및 $10^3$ SiHa 세포주에서 P<0.001를 나타내었고 $10^1$ 및 $10^2$ HeLa 세포주에서도 P<0.001를 나타내었다. 이를 통해 Takara 키트가 Invitrogen키트보다 민감도가 높음을 알 수 있었다. 또한 40개의 탈락세포검체의 8, 4, 2, 1 mL을 이용하여 상보적인 DNA 합성 키트를 비교한 결과 Invitrogen 키트보다 Takara 키트가 8, 4, 1 mL에서 P<0.01 및 0.5 mL에서 P<0.05을 나타내어 임상 검체를 이용하였을 때에도 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수 있었다. 본 연구는 적합한 상보적인 DNA 합성 키트를 확인하기 위해 수행되었으며, 상보적인 DNA 합성 키트가 real-time PCR 결과 다양성에 영향을 미친다는 것을 시사하였다.
Cervical cancer is the fourth most common malignant neoplasm in women worldwide. Most cases of cervical cancer are caused by an infection by the human papillomavirus. Molecular diagnostic methods have emerged to detect the HPV for sensitivity, specificity, and objectivity. In particular, real-time P...
Cervical cancer is the fourth most common malignant neoplasm in women worldwide. Most cases of cervical cancer are caused by an infection by the human papillomavirus. Molecular diagnostic methods have emerged to detect the HPV for sensitivity, specificity, and objectivity. In particular, real-time PCR has been introduced to acquire a more sensitive target DNA or RNA. RNA extraction and complementary DNA synthesis are proceeded before performing real-time PCR targeting RNA. To identify an adequate and sensitive cDNA synthesis kit, this study evaluated the two commonly used kits for cDNA synthesis. The results show that the $R^2$ and efficiency (%) of the two cDNA synthesis kits were similar in the cervical cancer cell lines. On the other hand, the Takara kit compared to Invitrogen kit showed P<0.001 in the $10^2$ and $10^3$ SiHa cell count. The Takara kit compared to the Invitrogen kit showed P<0.001 in the $10^1$ and $10^2$ HeLa cell count. Furthermore, 8, 4, 2, 1, and 0.5 ml of forty exfoliated cell samples were used to compare the cDNA synthesis kits. The Takara kit compared to the Invitrogen kit showed P<0.01 in 8, 4, and 1 ml and P<0.05 in 0.5 mL. The study was performed to identify the most appropriate cDNA synthesis kit and suggests that a cDNA synthesis kit could affect the real-time PCR results.
Cervical cancer is the fourth most common malignant neoplasm in women worldwide. Most cases of cervical cancer are caused by an infection by the human papillomavirus. Molecular diagnostic methods have emerged to detect the HPV for sensitivity, specificity, and objectivity. In particular, real-time PCR has been introduced to acquire a more sensitive target DNA or RNA. RNA extraction and complementary DNA synthesis are proceeded before performing real-time PCR targeting RNA. To identify an adequate and sensitive cDNA synthesis kit, this study evaluated the two commonly used kits for cDNA synthesis. The results show that the $R^2$ and efficiency (%) of the two cDNA synthesis kits were similar in the cervical cancer cell lines. On the other hand, the Takara kit compared to Invitrogen kit showed P<0.001 in the $10^2$ and $10^3$ SiHa cell count. The Takara kit compared to the Invitrogen kit showed P<0.001 in the $10^1$ and $10^2$ HeLa cell count. Furthermore, 8, 4, 2, 1, and 0.5 ml of forty exfoliated cell samples were used to compare the cDNA synthesis kits. The Takara kit compared to the Invitrogen kit showed P<0.01 in 8, 4, and 1 ml and P<0.05 in 0.5 mL. The study was performed to identify the most appropriate cDNA synthesis kit and suggests that a cDNA synthesis kit could affect the real-time PCR results.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
특히, real-time PCR 과정은 수행 전에 RNA 추출 및 상보적인 DNA 합성 과정이 필요하다. 따라서 본 연구에서는 민감하고 적합한 상보적인 DNA 합성 키트를 알아보기 위해서 상보적인 DNA 합성에 이용되는 두 개의 상용화된 키트를 평가하였다. 자궁경부암 세포주에서 두개의 상보적인 DNA 합성 키트의 R2과 효율성을 비교한 결과 차이가 없었다.
05을 나타내어 임상 검체를 이용하였을 때에도 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수있었다. 본 연구는 적합한 상보적인 DNA 합성 키트를 확인하기 위해 수행되었으며, 상보적인 DNA 합성 키트가 real-time PCR 결과 다양성에 영향을 미친다는 것을 시사하였다.
제안 방법
The enzyme of capacity is greater than any other commercial reverse transcriptase [35]. In the study, two commercial kits used RT originated from MMLV using cervical cancer cell lines and clinical samples.
In this study, we selected and compared two commercially available cDNA synthesis kits for the identification of the most useful kit in HPV screening test by cervical cancer cell lines and exfoliated cell samples. Reaction efficiency (%), coefficient of variation and cycle threshold (Ct) value analysis was performed.
The assay consists of three different sets of HR-HPV and detects 16 HR-HPV genotypes in three tubes (group I: HPV 16 [FAM], 33, 58 [HEX], and 31, 35 [Cy5]; group II: HPV 18 [FAM], 39, 68 [HEX], and 45∼51 [Cy5]; group III: HPV 53, 56, 66 [FAM], 59, 69 [HEX], and 52 [Cy5]), by incorporating specific TaqMan probes labeled with different fluorophores.
The purpose of the study was for evaluating the efficiency (%) of cDNA synthesis kits. For that study, HPV was selected to generate HPV RNA transcript.
대상 데이터
Cervical cancer cell lines (HeLa and SiHa) were purchased from American Type Culture Collection ATCC (Manassas, USA) and Korean Cell Line Bank (Seoul, Korea). SiHa and HeLa cell line were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Gibco, Carlsbad, USA), with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% streptomycin-penicillin (Gibco, Carlsbad, CA, USA).
The purpose of the study was for evaluating the efficiency (%) of cDNA synthesis kits. For that study, HPV was selected to generate HPV RNA transcript. We selected two different commercial cDNA synthesis kits for detecting HPV and evaluated the results using efficiency and R2 as shown in a paper [28].
All cell lines were incubated at 37°C in humidified 5% CO2 atmosphere. Forty exfoliated cell samples were collected from cervical cancer patients and healthy subjects at Wonju Severance Christian Hospital, Wonju, Korea, from January 2010 to December 2014. All subjects provided clinical information and this study was approved by the Institutional Ethics Committee of Yonsei University Wonju College of Medicine (Approval No.
성능/효과
Oligo dT has advantage that full-length cDNA can be synthesized and small amount of target mRNA can be amplified by several elongation steps by binding to same mRNA molecule. In conclusion, these results suggest quality of real-time PCR data analysis and experiment can be elevated according to cDNA synthesis kit strongly. Further studies need to be performed on a variety of clinical samples, more cDNA synthesis kits and various cancers.
96. Results demonstrated that there was no difference in reaction efficiency (%) and R 2 values between two methods in cervical cancer cell line which has HPV 16 and 18 for causing cervical cancer. Cervical cancer cell lines except other cell lines were used to define the diagnostic method for cervical cancer.
Results showed that efficiency (%) was between 90%∼110%, R2 was above 0.96.
이를 통해 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수 있었다. 또한 40개의 탈락세포검체의 8, 4, 2, 1 mL 을 이용하여 상보적인 DNA 합성 키트를 비교한 결과 Invitrogen 키트보다 Takara 키트가 8, 4, 1 mL에서 P＜0.01 및 0.5 mL에서 P＜0.05을 나타내어 임상 검체를 이용하였을 때에도 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수있었다. 본 연구는 적합한 상보적인 DNA 합성 키트를 확인하기 위해 수행되었으며, 상보적인 DNA 합성 키트가 real-time PCR 결과 다양성에 영향을 미친다는 것을 시사하였다.
001를 나타내었다. 이를 통해 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수 있었다. 또한 40개의 탈락세포검체의 8, 4, 2, 1 mL 을 이용하여 상보적인 DNA 합성 키트를 비교한 결과 Invitrogen 키트보다 Takara 키트가 8, 4, 1 mL에서 P＜0.
참고문헌 (37)
Bray, Freddie, Ferlay, Jacques, Soerjomataram, Isabelle, Siegel, Rebecca L., Torre, Lindsey A., Jemal, Ahmedin.
Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
CA: A Cancer Journal for Clinicians,
vol.68,
no.6,
394-424.
Jung, Kyu-Won, Won, Young-Joo, Kong, Hyun-Joo, Lee, Eun Sook.
Cancer Statistics in Korea: Incidence, Mortality, Survival, and Prevalence in 2015.
Cancer research and treatment : official journal of Korean Cancer Association,
vol.50,
no.2,
303-316.
Maucort-Boulch, Delphine, Franceschi, Silvia, Plummer, Martyn.
International Correlation between Human Papillomavirus Prevalence and Cervical Cancer Incidence.
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research,
vol.17,
no.3,
717-720.
Park SM, Lee SK, Kim YS. Inhibition of cervical cancer cell growth by gene silencing of HPV16 E6 induced by short-interfering RNA. Korean J Clin Lab Sci. 2011;43:89-97.
Baptista, Aimée Denzeler, Simão, Carolina Xavier, Santos, Vitoria Carvalho Guimarães dos, Melgaço, Juliana Gil, Cavalcanti, Silvia Maria Baeta, Fonseca, Sandra Costa, Vitral, Claudia Lamarca.
Knowledge of human papillomavirus and Pap test among Brazilian university students.
Revista da Associação Médica Brasileira,
vol.65,
no.5,
625-632.
Watson, Meg, Benard, Vicki, King, Jessica, Crawford, Anatasha, Saraiya, Mona.
National assessment of HPV and Pap tests: Changes in cervical cancer screening, National Health Interview Survey.
Preventive medicine,
vol.100,
243-247.
Al-Nourhji, Omar, Dermawan, Josephine Kamtai, Booth, Christine N., Underwood, Dawn, Abdul-Karim, Fadi W..
Role of ThinPrep liquid-based cytology in evaluation of the endocervical canal in patients with abnormal cervical screening.
Journal of the American Society of Cytopathology : JASC,
vol.8,
no.5,
278-283.
Koliopoulos, George, Nyaga, Victoria N, Santesso, Nancy, Bryant, Andrew, Martin-Hirsch, Pierre PL, Mustafa, Reem A, Schünemann, Holger, Paraskevaidis, Evangelos, Arbyn, Marc.
Cytology versus HPV testing for cervical cancer screening in the general population.
The Cochrane database of systematic reviews,
vol.2018,
no.7,
El-Zein, M., Richardson, L., Franco, E.L..
Cervical cancer screening of HPV vaccinated populations: Cytology, molecular testing, both or none.
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology,
vol.76,
no.suppl1,
S62-S68.
Poljak, M., Kocjan, B.J., Ostrbenk, A., Seme, K..
Commercially available molecular tests for human papillomaviruses (HPV): 2015 update.
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology,
vol.76,
no.suppl1,
S3-S13.
Depuydt, C E, Boulet, G A V, Horvath, C A J, Benoy, I H, Vereecken, A J, Bogers, J J.
Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.
Journal of cellular and molecular medicine,
vol.11,
no.4,
881-891.
Kim, Geehyuk, Park, Sungyoung, Wang, Hye-young, Kim, Sunghyun, Park, Sangjung, Yu, Kwangmin, Lee, Boohyung, Ahn, Seung-Ju, Kim, Eun-Joong, Lee, Dongsup.
Distribution of Oncogenic Human Papillomavirus Genotypes at High Grade Cervical Lesions above CIN 2 Grade with Histological Diagnosis.
Biomedical science letters = 대한의생명과학회지,
vol.22,
no.2,
37-45.
Roberts, Christine C., Swoyer, Ryan, Bryan, Janine T., Taddeo, Frank J..
Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with the Linear Array HPV Genotyping PCR Assay and Influence of DNA Extraction Method on HPV Detection.
Journal of clinical microbiology,
vol.49,
no.5,
1899-1906.
Pasquier, C., Saune, K., Raymond, S., Boisneau, J., Courtade, M., Izopet, J..
Comparison of Cobas® HPV and Anyplex(TM) II HPV28 assays for detecting and genotyping human papillomavirus.
Diagnostic microbiology and infectious disease,
vol.87,
no.1,
25-27.
Pan, Caixia, Zhou, Jing, Lyu, Jiangfeng, Ren, Xuyi.
Development and validation of a multiplex reverse transcript real-time PCR for E6/E7 mRNA detection of high-risk human papillomavirus.
Journal of medical microbiology,
vol.67,
no.10,
1509-1514.
Sahiner, F., Kubar, A., Yapar, M., Sener, K., Dede, M., Gumral, R..
Detection of major HPVs by a new multiplex real-time PCR assay using type-specific primers.
Journal of microbiological methods,
vol.97,
44-50.
Yang, Hui, Li, Lie-Jun, Xie, Long-Xu, Luo, Zhao-Yun, Lu, Min, Lin, Min, Zheng, Xiang-bin, Huang, Yue, Yang, Li-Ye.
Clinical validation of a novel real-time human papillomavirus assay for simultaneous detection of 14 high-risk HPV type and genotyping HPV type 16 and 18 in China.
Archives of virology,
vol.161,
no.2,
449-454.
Micalessi, Isabel M., Boulet, Gaëlle A.V., Bogers, Johannes J., Benoy, Ina H., Depuydt, Christophe E..
High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR.
Clinical chemistry and laboratory medicine : CCLM,
vol.50,
no.4,
655-661.
Harvey, R J, Darlison, M G.
Random-primed cDNA synthesis facilitates the isolation of multiple 5'-cDNA ends by RACE..
Nucleic acids research,
vol.19,
no.14,
4002-.
MICHAELIDOU, KLEITA, TZOVARAS, ALEXANDROS, MISSITZIS, IOANNIS, ARDAVANIS, ALEXANDROS, SCORILAS, ANDREAS.
The expression of the CEACAM19 gene, a novel member of the CEA family, is associated with breast cancer progression.
International journal of oncology,
vol.42,
no.5,
1770-1777.
Picard-Meyer, Evelyne, Peytavin de Garam, Carine, Schereffer, Jean Luc, Marchal, Clotilde, Robardet, Emmanuelle, Cliquet, Florence.
Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1.
BioMed research international,
vol.2015,
839518-.
Kim, G., Cho, H., Lee, D., Park, S., Lee, J., Wang, H.y., Kim, S., Park, K.H., Lee, H..
Comparison of FFPE histological versus LBP cytological samples for HPV detection and typing in cervical cancer.
Experimental and molecular pathology,
vol.102,
no.2,
321-326.
Wang, Hye-young, Lee, Dongsup, Park, Sunyoung, Kim, Geehyuk, Kim, Sunghyun, Han, Lin, Yubo, Ren, Li, Yingxue, Park, Kwang Hwa, Lee, Hyeyoung.
Diagnostic Performance of HPV E6/E7 mRNA and HPV DNA Assays for the Detection and Screening of Oncogenic Human Papillomavirus Infection among Woman with Cervical Lesions in China.
Asian Pacific journal of cancer prevention : APJCP,
vol.16,
no.17,
7633-7640.
Wang, H.y., Park, S., Lee, D., Kim, S., Kim, G., Park, K.H., Lee, H..
Prevalence of type-specific oncogenic human papillomavirus infection assessed by HPV E6/E7 mRNA among women with high-grade cervical lesions.
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases,
vol.37,
135-142.
Wacker MJ, Godard MP. Analysis of one-step and two-step real-time RT-PCR using SuperScript III. J Biomol Tech. 2005;16:266-271.
Jacob, Francis, Guertler, Rea, Naim, Stephanie, Nixdorf, Sheri, Fedier, André, Hacker, Neville F., Heinzelmann-Schwarz, Viola.
Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines.
PloS one,
vol.8,
no.3,
e59180-.
Pfaffl M. Quantification strategies in real-time PCR. A-Z of Quantitative PCR chapter, Bustin S. La Jolla, CA, USA: International University Line; 2004 p87-112.
10.1128/JVI.61.4.962-971.1987 Baker CC, Phelps WC, Lindgren V, Braun MJ, Gonda MA, Howley PM. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J Virol. 1987;61:962-971.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.