scholarly journals 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

2003 ◽  
Vol 31 (1) ◽  
pp. 123-132 ◽  
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.

Real-Time Reverse Transcriptase Polymerase Chain Reaction: An Improvement in Detecting mRNA Levels in Mouse Cranial Tissue

2006 ◽  
Vol 117 (7) ◽  
pp. 2227-2234
Author(s):  
Rashmi Singh ◽  
Ren?? F. Recinos ◽  
Michael Agresti ◽  
Richard B. Schaefer ◽  
Mark Bosbous ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Kuantifikasi Gen VDR pada Diabetes Melitus Tipe 2 dengan Metode Real-Time Polymerase Chain Reaction

2021 ◽  
Vol 7 (1) ◽  
pp. 1-8
Author(s):  
Fini Ainun Qolbi Wasdili ◽  
Sitti Romlah ◽  
Shintia Novianty
Keyword(s):  
Vitamin D ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Vitamin D Receptor ◽  
Real Time Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Diabetes Melitus

Diabetes melitus (DM) tipe 2 adalah penyakit yang ditandai dengan hiperglikemia akibat resistensi tubuh terhadap efek insulin yang diproduksi oleh sel beta pankreas. Pada lansia terjadi proses menua yang menyebabkan banyak perubahan, Defisiensi vitamin D dapat terjadi pada penderita DM tipe 2 dan mempercepat resistensi insulin. Vitamin D berperan pada imunomodulator yang melindungi terhadap kejadian DM melalui gen Vitamin D Receptor (VDR) yang diekspresikan pada sel penyaji antigen, sel T aktivasi dan sel β pankreas. Jumlah VDR yang ada di dalam tubuh tergantung pada gen VDR yang diekspresikan di dalam sel. Kuantifikasi peningkatan gen VDR pada lansia DM tipe 2 dengan metode Real-Time PCR. Penelitian ini menggunakan metode deskriptif laboratorik. Spesimen terdiri dari darah lansia yang menderita DM tipe 2 dan darah lansia dengan kadar glukosa normal. Ekstraksi DNA dari spesimen darah dengan metode spin kolom kemudian dilakukan amplifikasi menggunakan Real-Time PCR. Hasil Real-Time PCR dianalisis dengan menggunakan perhitungan rasio. Hasil penelitian nilai Ct normal adalah 21, sedangkan pada lansia penderita DM sangat bervariasi dengan nilai Ct paling kecil 15,22 dan nilai Ct paling tinggi 21,61. Disimpulkan terdapat variasi jumlah peningkatan gen VDR pada lansia penderita DM tipe 2.

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

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