Assessment of fHbp Expression Level by Reverse Transcriptase Quantitative PCR and Promoter Sequence Analysis

2019 ◽  
pp. 237-249
Author(s):  
Caroline Cayrou ◽  
Christopher D. Bayliss
Keyword(s):  
Sequence Analysis ◽  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Promoter Sequence ◽  
Expression Level

scholarly journals Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells.

1990 ◽  
Vol 10 (7) ◽  
pp. 3596-3606 ◽  
Author(s):  
S I Hsu ◽  
D Cohen ◽  
L S Kirschner ◽  
L Lothstein ◽  
M Hartstein ◽  
...  
Keyword(s):  
Steady State ◽  
Sequence Analysis ◽  
Protein Kinase ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Promoter Sequence ◽  
Multidrug Resistant ◽  
Transcriptional Unit ◽  
Resistant Mouse ◽  
Protein Levels

In multidrug-resistant mouse J774.2 cells, the differential overproduction of functionally distinct phosphoglycoprotein isoforms reflects the amplification or transcriptional activation or both of two mdr gene family members, mdr1a and mdr1b. The mdr1a gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes. Independently selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6- and 5.0-kilobase (kb) or 4.7- and 5.1-kb mdr1a transcripts. However, abundant overproduction of the mdr1a gene product was observed only in cell lines which overexpressed the 4.6- and 5.0-kb mRNAs. In order to determine the basis for mdr1a transcript heterogeneity and the relationship between transcript size and steady-state mdr1a protein levels, genomic and cDNA sequence analyses of the 5' and 3' ends of the mdr1a gene were carried out. Promoter sequence analysis and primer extension mapping indicated that mdr1a transcripts were differentially initiated from two putative promoters to generate either 5.1- and 4.7-kb or 5.0- and 4.6-kb transcripts in four multidrug-resistant J774.2 cell lines. Sequence analysis of 3' cDNA variants and a 3' genomic fragment revealed that the 5.1- and 5.0-kb mRNAs had identical 3'-untranslated regions which differed from those of the 4.7- and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal. Transcript initiation from the putative upstream promoter correlated with a 70 to 85% decrease in steady-state mdr1a protein levels relative to transcript levels. In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdr1a. The implications of these findings for mdr gene expression and regulation are discussed.

Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B

1995 ◽  
Vol 109 (5) ◽  
pp. 1618-1623 ◽  
Author(s):  
Tomasz Laskus ◽  
Jorge Rakela ◽  
Marek J. Nowicki ◽  
David H. Persing
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis B Virus ◽  
Sequence Analysis ◽  
Hepatitis B ◽  
Chronic Hepatitis B ◽  
Core Promoter ◽  
Promoter Sequence ◽  
Virus Core ◽  
B Virus ◽  
Core Promoter Sequence

scholarly journals Optimization of RNA Extraction for PCR Quantification of Aromatic Compound Degradation Genes

2009 ◽  
Vol 76 (4) ◽  
pp. 1282-1284 ◽  
Author(s):  
Weidong Kong ◽  
Cindy H. Nakatsu
Keyword(s):  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Aromatic Compound ◽  
Rna Extraction ◽  
Maximum Activity ◽  
Bacterial Strains ◽  
Aromatic Compound Degradation ◽  
Polluted Sites ◽  
Primer Sets ◽  
Mixed Community

ABSTRACT Seven different bacterial strains and primer sets and a mixed community were used to evaluate the use of reverse transcriptase quantitative PCR (Q-PCR) and Q-PCR of oxygenase genes to assess various approaches for monitoring the bioremediation of polluted sites. Differences in maximum activity were seen when different RNA extraction kits were compared.

Reverse Transcription with a Random Pentadecamer Primer Increases the Sensitivity of Quantitative PCR for BCR-ABL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2339-2339
Author(s):  
David M. Ross ◽  
Timothy P. Hughes ◽  
Susan Branford
Keyword(s):  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Negative Control ◽  
Gene Copy ◽  
Imatinib Treatment ◽  
Control Gene ◽  
Simple Modification ◽  
Number Of Patients

Abstract Real-time quantitative reverse transcriptase PCR for BCR-ABL (RQ-PCR) is used to monitor treatment response in chronic myeloid leukaemia (CML). BCR-ABL levels continue to decline over several years of imatinib treatment and increasing numbers of patients have BCR-ABL levels at or below the limit of detection. The sensitivity of current RQ-PCR assays limits our ability to identify patients who have a continuing decline in BCR-ABL levels; or to assess the effects of novel therapies to improve outcome in patients who have a good response to ABL kinase inhibitors, but harbour minimal residual disease. Improvements in therapy for these patients will be hard to assess without more sensitive monitoring of BCR-ABL. BCR-ABL levels are usually reported relative to a control gene. The control gene value gives an indication of the sensitivity achieved within each RNA sample; this varies with sample quality and efficiency of reverse transcription (RT). Control gene copy number below a defined value indicates that the analysis may be unreliable. We investigated the use of a random pentadecamer (RP; 15mer) primer in the RT reaction to improve the sensitivity of RQ-PCR. The RP primer is reported to be more efficient than the random hexamer (RH; 6mer) which is commonly used for RQ-PCR. BCR-ABL and BCR control gene transcripts were measured in 30 peripheral blood samples. After Trizol extraction 2μg RNA and 400U Superscript II reverse transcriptase were added to two RT reactions using RP or RH at a final concentration of 25μM. Each RT and quantitative PCR was performed 2–3 times and results compared using the Wilcoxon signed rank test or paired t-test. A more efficient RT is indicated by higher transcript levels. The table shows that BCR-ABL and BCR control gene values were significantly higher with RP primers. There was a proportionate increase in all transcripts so that the change in BCR-ABL/BCR% was not significant. BCR-ABL and control gene values with pentadecamer or hexamer primers No. of Samples Primer Median 25th Percentile 75th Percentile P value (RP v HP) * missing values due to inclusion of samples with undetectable BCR-ABL BCR transcripts 30 RH 438900 241800 1090000 < 0.001 RP 1203700 836500 2125000 BCR-ABL transcripts 16* RH 280 110 11340 < 0.001 RP 390 220 32910 BCR-ABL/BCR ratio 16* RH 0.10% 0.06% 5.6% =0.083 RP 0.08% 0.05% 4.2% We tested samples with undetectable BCR-ABL including 10 from CML patients on imatinib, and 10 from BCR-ABL negative control subjects. Each RNA was tested in two independent RTs and Q-PCRs. If the results were discordant a third RQ-PCR was performed and a consensus result determined. Using RP primers 6/10 patient samples were positive; 2/10 were positive with RH. None of the BCR-ABL negative control samples was positive with either primer. Sensitivity was calculated using the lower limit of detection and the standardised baseline value for untreated CML patients.The median calculated sensitivity increased from 4.5-log with RH to 5.0-log with RP. The use of pentadecamer primer made possible the detection of BCR-ABL in a small number of patients who had undetectable BCR-ABL with random hexamer RQ-PCR. This initial analysis suggests that the sensitivity of BCR-ABL detection may be improved 2–3-fold with a simple modification to RT methodology. This also has the potential to reduce the number of samples deemed unsatisfactory due to low control gene levels.

scholarly journals Importance of Micromonospora spp. as Colonizers of Cellulose in Freshwater Lakes as Demonstrated by Quantitative Reverse Transcriptase PCR of 16S rRNA

2012 ◽  
Vol 78 (9) ◽  
pp. 3495-3499 ◽  
Author(s):  
Alexandre B. de Menezes ◽  
James E. McDonald ◽  
Heather E. Allison ◽  
Alan J. McCarthy
Keyword(s):  
16S Rrna ◽  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Lake Sediment ◽  
Relative Abundance ◽  
Bacterial Communities ◽  
Bacterial Population ◽  
Freshwater Lakes ◽  
Content Type ◽  
Water Columns

ABSTRACTThe relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, water columns, and sediments of two freshwater lakes was determined by quantitative PCR (qPCR) of reverse-transcribed 16S rRNA.Micromonosporaspp. were shown to be significant members of the active bacterial population colonizing cellulosic substrates in the lake sediment, and their increased prevalence with greater depth was confirmed by enumeration of CFU.

TRES: comparative promoter sequence analysis

2000 ◽  
Vol 16 (8) ◽  
pp. 739-740 ◽  
Author(s):  
M. V. Katti ◽  
M. K. Sakharkar ◽  
P. K. Ranjekar ◽  
V. S. Gupta
Keyword(s):  
Sequence Analysis ◽  
Promoter Sequence

scholarly journals New inducers revealed by the promoter sequence analysis of two interferon-activated human genes

1987 ◽  
Vol 169 (2) ◽  
pp. 313-321 ◽  
Author(s):  
Marc G. WATHELET ◽  
Isabelle M. CLAUSS ◽  
Christine B. NOLS ◽  
Jean CONTENT ◽  
Georges A. HUEZ
Keyword(s):  
Sequence Analysis ◽  
Promoter Sequence ◽  
Human Genes

scholarly journals Role of Thrombospondin-1 in Sepsis-Induced Myocardial Injury

2021 ◽  
Author(s):  
YUN XIE ◽  
JIAXIANG ZHANG ◽  
WEI JIN ◽  
RUI TIAN ◽  
Ruilan Wang
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Myocardial Injury ◽  
Cell Injury ◽  
Myocardial Cell ◽  
Expression Level ◽  
Myocardial Cells ◽  
Thrombospondin 1 ◽  
Myocardial Cell Injury

Abstract Objective: Sepsis often causes myocardial injury with a high mortality. We wanted to investigate the effects of thrombospondin-1 (THBS1) expression on myocardial cell injury, oxidative stress and apoptosis in sepsis.Methods: The expression of THBS1 mRNA in LPS-induced mouse primary cardiomyocytes was detected by real-time fluorescence quantitative PCR. We constructed a eukaryotic siRNA expression vector and used liposome transfection to knockdown THBS1 mRNA expression in myocardial cells. We detected the THBS1 mRNA expression level using real-time fluorescent quantitative PCR. Four groups were used: control, LPS, THBS1 siRNA, and LPS + THBS1 siRNA. ELISA was used to detect cTnI, proBNP, ROS, caspase3 and other indicators of cell damage. At the same time, sepsis mouse models were prepared for H&E, TUNEL and caspase-3 staining to evaluate myocardial cell injury and apoptosis. Clinical samples were collected to analyze the serum THBS1 level and correlate it with the prognosis of patients with myocardial injury of sepsis.Results: The expression level of THBS1 mRNA in myocardial cells induced by LPS was increased, and the serum THBS1 level in patients with myocardial injury in sepsis was also significantly increased. In the THBS1 siRNA group with myocardial injury, the levels of cTnI and proBNP were significantly decreased, the levels of the inflammatory cytokines IL-6 and TNF-α were significantly decreased, ROS were significantly decreased, and caspase3 was significantly decreased, and myocardial cell apoptosis was also reduced in the sepsis mouse model. Conclusion: THBS1 is closely related to the biological behavior of myocardial cells and may be a therapeutic target for myocardial injury in sepsis.

scholarly journals Detection of SARS-CoV-2 in wastewater in Halifax, Nova Scotia, Canada, using four RT-qPCR assays

FACETS ◽  
2021 ◽  
Vol 6 ◽  
pp. 959-965
Author(s):  
Yannan Huang ◽  
Lindsay Johnston ◽  
Ana Parra ◽  
Crystal Sweeney ◽  
Emalie Hayes ◽  
...  
Keyword(s):  
Nova Scotia ◽  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Relative Sensitivity ◽  
Initial Rise ◽  
Specific Focus ◽  
Wastewater Samples

Wastewater-based surveillance methods have been implemented in several countries as a tool for monitoring SARS-CoV-2 at a community scale. A variety of methods have been used for concentrating, extracting, and detecting the virus, with no clear consensus on the most effective approach. In this note, we report preliminary findings from a study that is tracking SARS-CoV-2 in wastewater in Halifax, Nova Scotia, with a specific focus on the use of four reverse transcriptase quantitative PCR (RT-qPCR) assays for detecting the virus in wastewater. We were able to detect the virus in wastewater samples during the initial rise of cases in the Halifax region in early November 2020. Levels of the targeted SARS-CoV-2 gene fragments increased and fell in response to reported cases of COVID-19. The CDC N1 and E RT-qPCR assays demonstrated greater relative sensitivity than the CDC N2 and N3 assays for detection of SARS-CoV-2 in raw sewage samples.

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