Real–Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT–PCR) Analysis of the Vitamin D Pathway in UV Irradiated Keratinocytes

Purpose To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream. Patients and Methods In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma ≥ 1.0 mm Breslow thickness, and no clinical evidence of metastasis. SLN biopsy and wide excision of the primary tumor were performed. SLNs were examined by serial-section histopathology and S-100 immunohistochemistry. A portion of each SLN was frozen for RT-PCR. In addition, RT-PCR was performed on peripheral-blood mononuclear cells (PBMCs). RT-PCR analysis was performed using four markers: tyrosinase, MART1, MAGE3, and GP-100. Disease-free survival (DFS), distant–DFS (DDFS), and overall survival (OS) were analyzed. Results A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR–positive (n = 620) and RT-PCR–negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood. Conclusion In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.

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REAL TIME POLYMERASE CHAIN REACTION (RT-PCR) FOR DNA PIG CONTAMINATION IDENTIFICATION SAUSAGE IN SIX DISTRICT PANDEGLANG BANTEN

International Journal Mathla’ul Anwar of Halal Issues ◽

10.30653/ijma.202111.13 ◽

2021 ◽

Vol 1 (1) ◽

pp. 81-88

Author(s):

Hadi Susilo

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Meat Product ◽

Meat Products ◽

Pcr Analysis ◽

Processed Meat ◽

Rt Pcr ◽

Chain Reaction ◽

Dna Purity ◽

Polymerase Chain

Sausage is a meat product processed that is popular food especially in Pandeglang, Banten Province. The importance of halal certificates or the existence of the MUI (Indonesian Ulama Council) halal logo for processed meat products makes Muslim people confident to consume them. The aim this research was to identify pig DNA contamination in sausage products in six districts in Pandeglang without the MUI halal labels using RT-PCR (Real Time-Polymerase Chain Reaction). RT PCR that can calculate to pig to fill these sample free from pig contamination. This research was divided into two stage, the first stage is extracted or carried out DNA and the second stage is RT PCR analysis. The results of the DNA purity test on sausage samples had DNA purity values of 1.84-1.9 (A260 / A280) and resulted in sample concentrations ranging from 37.8 to 102.5 ng / µl. The only amplification on the FAM curve was in the positive control pig. the Cq value ranges from 30 - 31.29. The results of RT PCR on sausage samples in the district area in Pandeglang Banten did not detect the presence of pig DNA.

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Optimization of Real-time Reverse Transcriptase Polymerase Chain Reaction for Detection of Dengue Virus

Journal of Advances in Microbiology ◽

10.9734/jamb/2020/v20i830269 ◽

2020 ◽

pp. 1-7

Author(s):

Kaunara A. Azizi ◽

Arnold J. Ndaro ◽

Athanasia Maro ◽

Adonira Saro ◽

Reginald A. Kavishe

Keyword(s):

Polymerase Chain Reaction ◽

Nucleic Acid ◽

Dengue Virus ◽

Reverse Transcriptase ◽

Real Time ◽

Acid Extraction ◽

Rt Pcr ◽

Chain Reaction ◽

Reliable Technique ◽

Polymerase Chain

Aims: This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method. Methodology: One step and two step real time RT-PCR were evaluated in different PCR thermocyclers. Extraction of viral RNA was done by using a simple boom method. Results: The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

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Improved sensitivity for detecting micrometastases in pelvic lymph nodes by real-time reverse transcriptase polymerase chain reaction (RT-PCR) compared with conventional RT-PCR in patients with clinically localized prostate cancer undergoing radical prostatectomy

BJU International ◽

10.1111/j.1464-410x.2008.08322.x ◽

2009 ◽

Vol 103 (8) ◽

pp. 1074-1078 ◽

Cited By ~ 14

Author(s):

Tomoaki Terakawa ◽

Hideaki Miyake ◽

Toshifumi Kurahashi ◽

Junya Furukawa ◽

Atsushi Takenaka ◽

...

Keyword(s):

Prostate Cancer ◽

Polymerase Chain Reaction ◽

Radical Prostatectomy ◽

Lymph Nodes ◽

Reverse Transcriptase ◽

Real Time ◽

Rt Pcr ◽

Chain Reaction ◽

Pelvic Lymph Nodes ◽

Polymerase Chain

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Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310123 ◽

2003 ◽

Vol 31 (1) ◽

pp. 123-132 ◽

Cited By ~ 50

Author(s):

PH Anderson ◽

PD O'Loughlin ◽

BK May ◽

HA Morris

Keyword(s):

Vitamin D ◽

Polymerase Chain Reaction ◽

Reverse Transcriptase ◽

Real Time ◽

Vitamin D Receptor ◽

Mrna Levels ◽

Chain Reaction ◽

Hydroxyvitamin D ◽

25 Hydroxyvitamin D ◽

Polymerase Chain

Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.

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