Expression difference of miR-10b and miR-135b between the fertile and infertile semen samples (p)
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Semen Analysis
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Objectives To develop and evaluate a real-time fluorescent quantitative RT-PCR method for the detection of a novel CXC chemokine macrophage inflammatory protein-2γ(MIP-2γ)mRNA expression based on TaqMan technique.Method The expression of MIP-2γ mRNA was detected by TaqMan real time fluorscet quantitative RT-PCR. Results The dynamic range of the assay varied from 102 to 107 copies. The intra- and inter-assay coefficient variation (CV) were less than 122%. MIP-2γ mRNA is expressed in heart, liver and kidney of normal Balb/c mice, with the greatest expression in heart.Conclusion The detection of MIP-2γ mRNA expression by real time fluorescent quantitative RT-PCR based on TaqMan technique is more accurate,specific, sensitive and time-saved, which is suitable for use in clinical laboratory.
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Coefficient of variation
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Objective To establish a real-time quantitative RT-PCR method for detection of HLA-DRα mRNA expression.Methods The vector containing HLA-DRα gene as standard template was constructed by T-A cloning technique.A real-time RT-PCR was established by used the fluorogenic probe(Taqman probe).HLA-DRα mRNA expression level in monocytes induced by achyrathes bidentata polysacchari-(des(ABPS)) was detected with real-time quantitative RT-PCR and semi-quantitative RT-PCR.Results The expression of HLA-DRα mRNA in monocytes induced by ABPS was increased significantly,but no significant change of HLA-DRα mRNA expression was fund in control group;The semi-quantitative RTPCR results also demonstrated the variety of HLA-DRα level as what the real-time fluorogenic quantitative RT-PCR detected,but less sensitive and accurate.Conclusion The fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR in Detection of HLA-DRα mRNA expression.
TaqMan
Quantitative Analysis
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Objective To establish a real-time quantitative RT-PCR method for the detection of expression of TNF-α mRNA.Methods The vector containing TNF-α gene as standard template was constructed with T-A cloning technique.The fluorogenic probe(i.e,Taqman probe)was used to establish a real-time RT-PCR.TNF-α mRNA expression level in human peripheral blood mononuclear cells(PBMC) for tumor patients and normal persons was determined with real-time quantitative RT-PCR,also with semi-quantitative RT-PCR.Results The expression of TNF-α mRNA in PBMC for tumor patients was more high than that of normal persons;The semi-quantitative RT-PCR results also demonstrated that TNF-α level varied as detected with real-time fluorogenic quantitative RT-PCR,but less sensitive and accurate.Conclusion Detection of TNF-α mRNA expression with the fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR.
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Quantitative Analysis
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TaqMan
White spot syndrome
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Objective To compare the sensitivity, specificity and time-spending of TaqMan real-time RT-PCR and conventional RT-PCR for detecting dengue virus. Methods The real-time RT-PCR and the conventional RT-PCR assays were used to quantitatively detect dengue virus, Japanese encephalitis virus and West Nile virus and the sensitivity, specificity and time-spending were evaluated between the two methods above. Results Both Japanese encephalitis virus and West Nile were not detected by those two methods, however, dengue virus could be detected by both two methods. The sensitivity of the conventional RT-PCR was 0.1 TCID50/mL, while TaqMan real-time quantitative RT-PCR was 0.001TCID50/mL, which was 100 times sensitive than conventional RT-PCR. Meanwhile, the real-time RT-PCR was more time-saving than conventional RT-PCR. Conclusion TaqMan quantitative real-time RT-PCR assay is more sensitive and time-saving than conventional RT-PCR for the rapid diagnosis of dengue virus.
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Background: Infertility is both a clinical and a public problem. Standard semen analysis is the surrogate measure of male fertility in clinical practiceto determine prevalence of low sperm count including oligozoospermia and azoospermia and to assess the pattern and distribution of abnormal semen parameters in infertile men.Methods: The retrospective study was conducted with compiling of the data from archival record over a period of three years from June 2013 to June 2016. A total of 933 male partners of women attending the fertility clinic of hospital between the ages of 20 and 50 years were recruited. The samples taken were primary infertility cases using simple random sampling 4 technique. Semen analysis was performed according to the standards outlined by the World Health Organization (5thedition 2010). Parameters outlined included: Appearance, Volume,pH, Sperm concentration, Motility, Morphology, Viability and White cell countResult: Out of 933 samples; normozoospermia was observed in 659(70.6%) males, oligozoospermia 170(18.2%), and azoospermia 104(11.1%). The azoospermic and oligozoospermic samples had low ejaculated volume, but significantly higher percentage of pus cells in comparison to normozoospermic samples. The oligozoospermic samples had higher percentage of immotile sperms and abnormal morphology in comparison to normozoospermic samples. Asthenozoospermia was observed in 118(14.2%), teratozoospermia in 24(2.9%), and oligoteratozoospermia in 11(1.3%) of samples.Conclusion: Majority of cases of infertility in males show normal sperm count. Oligozoospermia followed by azoospermia is seen in rest of the cases while less sperm motility or less amount of semen are also responsible in some cases.DOI:10.21276/APALM.1502
Asthenozoospermia
Semen Analysis
Oligospermia
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Aim To optimize the taqman real-time PCR reaction system for quantitative detection of the expression of MSRG mRNA and evaluate the results.Methods Specific primers and probes were designed and real-time PCR was used for d etection of cDNA sequence.Taqman real-time PCR reaction system from template,primer,probe and Mg~(2+) concentration were optimized.Results Quantity of template was 2μl.The concentratons of primers,probes and Mg~(2+) were 0.4 μmol/L,0.2 μmol/L and 4mM.Conclusion Via optimizing the real-time PCR assay a specific,reliable tool for detecting MRSG mRNA expression levels.was established.
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Primer (cosmetics)
Primer dimer
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مقدمه و هدف: بروسلوز یک
بیماری مشترک بین انسان و دام ویک مشکل بهداشت عمومی در سراسر جهان می
باشد.بروسلوز باعث سقط جنین ،اختلالات باروری ،عفونت های تناسلی،کاهش شیر،اورتریت
و اپیدیدیمیت در میزبان اصلی می شود و از این جهت باعث عوارض مزمن و تحمیل هزینه
های درمانی فراوان به بیماران و ضررهای اقتصادی زیاد به دامداران می گردد.این بیماری،در تمام نقاط ایران پراکنده
است و ایران یکی ازمناطق آندمیک آلودگی به تب مالت است. شناسایی صحیح و سریع عامل
بیماری یکی ازعوامل مهم جهت کنترل و پیشگیری بیماری می باشد.با وجود پیشرفت هایی
که در تکنیک های کشت خون و تست های سرولوژیکی که برای کشف آنتی بادی های اختصاصی
به دست امد،هنوز مشکلات مهمی در تشخیص بروسلوزیس وجود دارد بنابراین نیاز به ازمون
های جدید آزمایشگاهی می باشد.یکی از جدیدترین روش های سنجش کمی که در حال حاضرمورد
توجه بسیاری از محققین قرار گرفته همین روش ها ی تشخیص محصول PCR در زمان
واقعی(Real Time) است.هدف ما هم از این مطالعه بهینه سازی
شناسایی باکتریهای جنس بروسلا در نمونه های مشکوک از نظر سرولوژیکی بوسیله روش Taqman Real-Time PCR می باشد. مواد و روش ها: 45 نمونه مثبت
(خون گوسفند) از نظر تستهای سرولوژیکی از چندین شهرستان در استان مازندران در طی 3
ماه تهیه گردید.پس از استخراج DNA نمونه ها با استفاده ازکیت MBST،واکنش Real-Time PCR TaqManبراساس
ژن bcsp31 و با استفاده از دستگاه Corbett 6000 روی نمونه ها انجام
گرفت. نتایج و بحث: از 45 نمونه مشکوک تنها 20 نمونه از نظر حضور باکتریهای
جنس بروسلا با استفاده از روشReal-Time PCR TaqMan مثبت بودند. تشخیص بروسلوز بواسطه شباهت
علائم بالینی آن با بسیاری از بیماری ها و عدم وجود روش های صحیح تشخیصی اغلب دشوار است.یکی ازروشهای دقیق که میتواند
مقادیر جزئی باکتری را شناسایی نماید،روشهایReal-Time PCR TaqMan می باشد که در آن علاوه بر یک جفت
پرایمراختصاصی،یک پروب اختصاصی نشاندار شده با ماده فلورسنت اختصاصیت روش را به بالاترین
میزان خود رسانده است. همچنین به دلیل حذف Post-PCR در تکنیک Real-Time PCR امکان وقوع Carry over و پاسخ مثبت کاذب نیز منتفی می گردد. مطالعه ما نشان می دهد که بخش زیادی از نمونه
های مشکوک به بروسلوزیس در حقیقت فاقد عفونت با جنس بروسلاهستند.
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The probes and primers were designed and synthesized according to the conserved gene ORF7 of PRRSV available in GenBank,and then reaction parameters were optimized to develop a real-time TaqMan-quantitative RT-PCR assay.The serum and other tissue samples from pigs on farms in Dongguan,Zengcheng,Zhanjiang and other areas of Guangdong Province were detected by using the established quantitative RT-PCR assay,and the results was compared with that of routine RT-PCR.The developed quantitative RT-PCR assay could detect 5.0×10~(0) copy/μL of plasmid DNA and its sensitivity was 100 times higher than that of the routine RT-PCR,while the results of the quantitative RT-PCR were the same as that of the routine RT-PCR.Three samples were examined using the real-time RT-PCR repeatedly and the results indicated that the real-time quantitative RT-PCR was reproducible and could be used for the(diagnosis) of PRRSV infection.
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We establish a TaqMan real-time PCR assay for detection of PRRSV.The specific primers and probes were designed in the conserved region of the ORF7 gene for PRRSV,and the real-time fluorescent quantitative PCR was established by optimizing the probe concentration.Thirty clinical samples were detected by using the established quantitative RT-PCR assay,and the results were compared with that of conventional RT-PCR and viral isolation tests.TaqMan fluorescent quantitative PCR for detection of PRRSV was established successfully with the optimal probe concentration 0.4 μmol,and detection limit was as low as 3.51 copies/μl.The results by the TaqMan real-time PCR method were 100% consistent with the viral isolation tests.Sensitivity and positive rate(28/30) for clinical samples of TaqMan fluorescent quantitative PCR were relatively higher than conventional PCR(25/30).The results indicated this method has high specificity,sensitivity and reproducibility,and could be used for the diagnosis of PRRSV infection.
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