scholarly journals PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation

2003 ◽  
Vol 49 (3) ◽  
pp. 425-432 ◽  
Author(s):  
Reidun Øvstebø ◽  
Kari Bente Foss Haug ◽  
Knut Lande ◽  
Peter Kierulf
Keyword(s):  
Total Variation ◽  
Reverse Transcription ◽  
Target Gene ◽  
Concentration Difference ◽  
Reverse Transcription Pcr ◽  
Calibration Curves ◽  
Reverse Transcription Step ◽  
Actin Mrna ◽  
Stringent Quality ◽  
Specific Mrna

Abstract Background: Quantitative reverse transcription-PCR (RT-PCR) used to detect small changes in specific mRNA concentrations is often associated with poor reproducibility. Thus, there is a need for stringent quality control in each step of the protocol. Methods: Real-time PCR-based calibration curves for a target gene, tissue factor (TF), and a reference gene, β-actin, generated from PCR amplicons were evaluated by running cDNA controls. In addition, the reverse transcription step was evaluated by running mRNA controls. Amplification efficiencies of calibrators and targets were determined. Variances within and between runs were estimated, and power statistics were applied to determine the concentration differences that could reliably be detected. Results: Within- and between-run variations (CVs) of cDNA controls (TF and β-actin), extrapolated from reproducible calibration curves (CVs of slopes, 4.3% and 2.7%, respectively) were 4–10% (within) and 15–38% (between) using both daily and “grand mean” calibration curves. CVs for the β-actin mRNA controls were 12% (within) and 19–28% (between). Estimates of each step’s contribution to the total variation were as follows: CVRT-PCR, 28%; CVPCR, 15%; CVRT, 23% (difference between CVRT-PCR and CVPCR). PCR efficiencies were as follows: β-actin calibrator/target, 1.96/1.95; TF calibrator/target, 1.95/1.93. Duplicate measurements could detect a twofold concentration difference (power, 0.8). Conclusions: Daily PCR calibration curves generated from PCR amplicons were reproducible, allowing the use of a grand mean calibration curve. The reverse transcription step contributes the most to the total variation. By determining a system’s total variance, power analysis may be used to disclose differences that can be reliably detected at a specified power.

scholarly journals Diagnosis and Molecular Monitoring of Acute Promyelocytic Leukemia Using DzyNA Reverse Transcription-PCR to Quantify PML/RARα Fusion Transcripts

2002 ◽  
Vol 48 (8) ◽  
pp. 1338-1343 ◽  
Author(s):  
Tanya L Applegate ◽  
Harry J Iland ◽  
Elisa Mokany ◽  
Alison V Todd
Keyword(s):  
Acute Promyelocytic Leukemia ◽  
Reverse Transcription ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Response To Therapy ◽  
Rt Pcr ◽  
Molecular Monitoring ◽  
Fusion Transcripts ◽  
Reverse Transcription Pcr ◽  
Calibration Curves

Abstract Background: PML/RARα fusion transcripts provide a readily accessible marker for diagnosis of acute promyelocytic leukemia (APL) and for monitoring response to therapy. Survival rates are improved by therapies guided by such monitoring. We assessed the potential of DzyNA reverse transcription-PCR (RT-PCR) for measurement of PML/RARα fusion transcripts. Methods: Parallel single-tube DzyNA RT-PCR protocols were developed to allow real-time fluorescent quantification of PML/RARα fusion transcripts and a low abundance control transcript, normal BCR. Calibration curves, generated using cell line RNA, allowed estimation of these transcripts in RNA from patients with APL at various stages of the disease. Results: DzyNA RT-PCR calibration curves were linear for both transcripts over a broad range and demonstrated interassay variations of 12% (mean, 658 ng) and 10% (mean, 263 ng), respectively. The protocols detected low concentrations of transcripts and resolved twofold dilutions. PML/RARα mRNA was quantified in 10 patients at diagnosis and in 1 patient over a 7-year period. Monitoring of transcript concentrations effectively reflected the disease course in one patient and demonstrated that an increase in PML/RARα transcripts can be detected 4–6 months before hematologic relapse, with no false-positive results. Conclusion: DzyNA RT-PCR has potential for use in clinical practice as a tool for diagnosis of APL and forsubsequent monitoring of minimal residual disease and detection of molecular relapse.

scholarly journals Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR

2013 ◽  
Vol 14 (1) ◽  
pp. 1093-1104 ◽  
Author(s):  
Ulrich Andergassen ◽  
Simone Hofmann ◽  
Alexandra Kölbl ◽  
Christian Schindlbeck ◽  
Julia Neugebauer ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Real Time ◽  
Reverse Transcription ◽  
Peripheral Blood ◽  
Reverse Transcription Pcr ◽  
Specific Mrna

scholarly journals Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

2021 ◽  
pp. 104868
Author(s):  
Marielle BEDOTTO ◽  
Pierre-Edouard FOURNIER ◽  
Linda HOUHAMDI ◽  
Philippe COLSON ◽  
Didier RAOULT
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

scholarly journals Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg
Keyword(s):  
Reverse Transcription ◽  
Membrane Filtration ◽  
Limit Of Detection ◽  
Mineral Waters ◽  
Pcr Analysis ◽  
Rna Sequences ◽  
Natural Mineral ◽  
Reverse Transcription Pcr ◽  
Viral Particles ◽  
Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

Direct subtyping of HLA-B and -A genes by automated sequencing following reverse transcription-PCR

1996 ◽  
Vol 47 (1-2) ◽  
pp. 115
Author(s):  
AJ Knipper ◽  
J Enczmann ◽  
P Hakenberg ◽  
G Kögler ◽  
P Wernet
Keyword(s):  
Reverse Transcription ◽  
Reverse Transcription Pcr

scholarly journals Aromatic Hydroxylation of Indan by o-Xylene-Degrading Rhodococcus sp. Strain DK17

2009 ◽  
Vol 76 (1) ◽  
pp. 375-377 ◽  
Author(s):  
Dockyu Kim ◽  
Choong Hwan Lee ◽  
Jung Nam Choi ◽  
Ki Young Choi ◽  
Gerben J. Zylstra ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Ring Cleavage ◽  
Growth Substrate ◽  
Aromatic Moiety ◽  
Reverse Transcription Pcr ◽  
Aromatic Hydroxylation ◽  
Rhodococcus Sp

ABSTRACT The metabolically versatile Rhodococcus sp. strain DK17 utilizes indan as a growth substrate via the o-xylene pathway. Metabolite and reverse transcription-PCR analyses indicate that o-xylene dioxygenase hydroxylates indan at the 4,5 position of the aromatic moiety to form cis-indan-4,5-dihydrodiol, which is dehydrogenated to 4,5-indandiol by a dehydrogenase. 4,5-Indandiol undergoes ring cleavage by a meta-cleavage dioxygenase.

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