A Quantitative Multistandard Reverse Transcriptase–Polymerase Chain Reaction Assay of the Cystic Fibrosis Transmembrane Conductance Regulator: Its Usefulness in Studying Efficiency of Gene Transfer

2000 ◽  
Vol 283 (2) ◽  
pp. 200-206 ◽  
Author(s):  
Stéphanie Marchand-Pinatel ◽  
Richard Planells ◽  
Marc D. Merten ◽  
Wafa Kammouni ◽  
Catherine Figarella
Keyword(s):  
Cystic Fibrosis ◽  
Polymerase Chain Reaction ◽  
Gene Transfer ◽  
Reverse Transcriptase ◽  
Polymerase Chain Reaction Assay ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Cystic Fibrosis Transmembrane

scholarly journals AB0691 ANALYSIS OF SARS-COV-2 ANTIBODIES IN NON-COVID-19 PATIENTS: COMPARISON BETWEEN SYSTEMIC SCLEROSIS PATIENTS AND HEALTHY CONTROLS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1379.1-1379
Author(s):  
L. Giardullo ◽  
C. Rotondo ◽  
A. Corrado ◽  
N. Maruotti ◽  
R. Colia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Systemic Sclerosis ◽  
Autoimmune Disease ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Nuclear Antigen ◽  
Healthy Controls ◽  
Chain Reaction ◽  
Polymerase Chain

Background:Previous study evidenced a cross-reactivity between Sars-Cov-2 antibodies and autoimmune tissue antigen involved in connective tissue diseases, as nuclear antigen (NA), extractable nuclear antigen (ENA), histone and collagen (1). No study has been published about the titer of Sars-Cov-2 antibodies in non-infected patients with autoimmune disease.Objectives:To evaluate the titer of SARS-CoV-2 antibodies in non-COVID-19 patients and compare it between systemic sclerosis (SSc) patients and healthy controls (HC).Methods:A total of 58 patients with SSc (who fulfilled ACR/EULAR 2013 SSc classification criteria) and 18 HC were enrolled. Sera of all participants were collected, and SARS-CoV-2 antibodies (IgG and IgM) were evaluated by means ELISA. In all participants swabs for SARS-CoV-2 by real-time reverse-transcriptase-polymerase-chain-reaction assay were reported negative. Demographic, clinical, and autoimmune serological characteristics of SSc patients were recorded. The normal distribution was assessed using the Shapiro–Wilk’s test. Exclusion criteria was previous or actual Sars-Cov-2 infection. Comparisons between study groups of patients were evaluated by the Student’s t-test or Mann – Whitney U-test as appropriate. The differences between categorial variables were assessed by Pearson chi-square or Fisher’s exact test, as opportune. Statistical significance was set at p ≤ 0.05.Results:We observed significant differences between SSc patients and HC in serum levels of Sars-Cov-2 antibodies (IgG: 1,4±2,1 AU/ml vs 0,36±0,19 AU/ml respectively (p=0,001); and IgM: 2,5±3,1 AU/ml vs 0,8±0,7 AU/ml (p=0,022)). In 5 SSc patients was found titer of Sars-Cov-2 antibodies (IgG) exceeding the cut-off, but the control of swabs for SARS-CoV-2 by real-time reverse-transcriptase-polymerase-chain-reaction assay were negative. No significative differences in Sars-Cov-2 autoantibodies titer were found in subgroup of SSc patients with or without ILD or PAH, limited or diffuse skin subset, and different autoantibodies profile. Furthermore, antibodies titer was not associated with different drugs (steroid, methotrexate, mofetil-mycophenolate and bosentan) in use.Conclusion:A cross mimicking between Sars-Cov-2 antibodies and antinuclear antibodies or anti ENA could be hypothesized. Further studies are necessary to unravel the reliability of Sars-Cov-2 antibodies detection in autoimmune disease.References:[1]Vojdani, A., Vojdani, E., & Kharrazian, D. (2021). Reaction of human monoclonal antibodies to SARS-CoV-2 proteins with tissue antigens: Implications for autoimmune diseases. Frontiers in Immunology, 11, 3679Disclosure of Interests:None declared

scholarly journals Competitive RT-PCR to quantify CFTR mRNA in human endometrium

1996 ◽  
Vol 42 (11) ◽  
pp. 1765-1769 ◽  
Author(s):  
A Mularoni ◽  
G L Adessi ◽  
F Arbez-Gindre ◽  
G Agnani ◽  
M Nicollier
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Specific Marker ◽  
Transmembrane Conductance Regulator ◽  
Rt Pcr ◽  
Transmembrane Conductance ◽  
Chain Reaction ◽  
Mrna Concentration ◽  
Nucleotide Insertion ◽  
Polymerase Chain ◽  
Cystic Fibrosis Transmembrane

Abstract Because the down-regulation by progesterone of cystic fibrosis transmembrane conductance regulator (CFTR) expression could be a useful specific marker to define the state of implant receptivity in endometrium, a competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed for quantifying the CFTR mRNA concentration in human endometrial samples. A competitor RNA was constructed with the same sequence as the CFTR sequence except for a 20-nucleotide insertion in the middle. The amplified products were separated by polyacrylamide gel electrophoresis. The ratio of CFTR band areas to competitor band areas provided the basis of quantification. Using this competitive RT-PCR, we measured CFTR mRNA in human endometrial samples taken at different periods of the menstrual cycle, in endometriosis, and in hyperplasia. Results show that the method is suitable for measuring the concentration of CFTR mRNA.

Cloning and sequence analysis of rat cystic fibrosis transmembrane conductance regulator

1992 ◽  
Vol 262 (6) ◽  
pp. L779-L784 ◽  
Author(s):  
M. A. Fiedler ◽  
Z. K. Nemecz ◽  
G. E. Shull
Keyword(s):  
Cystic Fibrosis ◽  
Northern Blot Analysis ◽  
Polymerase Chain Reaction Amplification ◽  
Rat Colon ◽  
Predicted Amino Acid Sequence ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Reaction Amplification ◽  
Polymerase Chain ◽  
Cystic Fibrosis Transmembrane

A complementary DNA (cDNA) encoding the rat cystic fibrosis transmembrane conductance regulator (CFTR) has been isolated and the tissue distribution of the rat CFTR mRNA has been determined. Northern blot analysis revealed that the highest levels of the 6.3 kilobase (kb) CFTR mRNA were expressed in the colon, with expression also noted in uterus, lung, stomach, and small intestine. A 7.5-kb mRNA was expressed in skeletal muscle, and in testes both the 7.5-kb mRNA and a 6.0-kb mRNA were expressed. Five cDNAs were isolated from a rat colon library, the longest corresponding to codons 684 through the poly A tail. Three other clones, corresponding to codons 213 through 245, 372 through 574, and 656 through 886 were also isolated. Polymerase chain reaction amplification of cDNA prepared from rat colon mRNA was utilized to clone the remainder of the cDNA. The predicted amino acid sequence of the rat CFTR is 79% identical to the human CFTR, with 73% identity noted in the R domain, and 81 and 83% identities noted in nucleotide binding folds 1 and 2, respectively. Thirty-two of the 38 potential phosphorylation sites identified in the human CFTR were also present in the rat CFTR.

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