Computational protocol: Tumor Necrosis Factor-Superfamily 15 Gene Expression in Patients with Sickle Cell Disease

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Protocol publication

[…] Study Population We investigated TNFSF15 gene expression and serum levels in children with SCD. The diagnosis of SCD was confirmed according to hematological and clinical data. Forty-nine children (34 males and 15 females; mean age: 9.24±3.66 years, range: 3-17 years) with SCD (38 SS, 11 Sβ), who were followed in the Pediatric Hematology Unit of Mersin University, Mersin, Turkey, were included in the study (Group 1: patients with SCD). Thirty-eight children (19 males and 19 females; mean age: 7.84±3.53 years, range: 2-16 years) were consecutively selected from healthy unrelated subjects from the same geographic area of Turkey (Group 2; controls).The information about painful crisis, acute chest syndrome (ACS), and stroke was taken from hospital medical records.Patients who had histories of vaso-occlusive crises and erythrocyte transfusion in the last 1 month were excluded.The patients were classified according to history of painful crises in the last 1 year. Group 1a included patients who had no painful crises, Group 1b included patients who had 1-4 painful crises, Group 1c included patients who had 5-10 painful crises, and Group 1d included patients who had more than 10 painful crises in the last 1 year. The patients were also classified according to ACS data.The protocol of this study was approved by the Ethics Committee of the School of Medicine of Mersin University. The investigation conforms to the principles outlined in the Declaration of Helsinki. RNA Isolation and Quantitative Real-Time PCR Blood samples were collected from patients with SCD who had been treated at Mersin University Research Hospital and from healthy subjects as controls. The blood mononuclear cells were separated by Red Blood Cell Lysis Buffer (Roche, Mannheim, Germany). Total RNA was extracted from peripheral blood mononuclear cells using the High Pure RNA Isolation Kit (Roche) following the manufacturer’s protocol including an additional genomic DNA digestion step with DNase I.Five micrograms of total RNA was used to synthesize cDNA. First-strand cDNA synthesis was conducted using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s recommendations. Real-time monitoring of PCR reactions was performed with the LightCycler®2.0 Real-Time PCR Detection System (Roche). Each reaction was conducted with TNFSF15-specific primers. The specific PCR assay was designed with the web-based Probe Finder software, using primers and probes from the Universal Probe Library (www.roche-applied-science.com/sis/rtpcr/upl/index.jsp, UPL, Roche). We selected a UPL assay for the TNFSF15 real-time PCR with the following primer-probe combination: forward primer 5′- caagggcacacctgacagt -3′ and reverse primer 5′- cctagttcatgttcccagtgc- 3′, with UPL probe #33 (Cat. No. 04687663001, Roche). β-Actin (ACTB) was used as an internal control (Cat. No. 0504616500, UPL Human ACTB Gene Assay, Roche). Determination of Serum TNFSF15 Serum TNFSF15 levels were determined by ELISA assay by pairing a monoclonal antibody with biotinylated anti-TNFSF15. A monoclonal antibody specific for hTL1A was coated onto the wells of the microtiter plate. hTL1A was recognized by the addition of a biotinylated monoclonal antibody specific for hTL1A. After removal of excess biotinylated antibody, streptavidin-peroxidase was added. Peroxidase activity was quantified using the substrate 3,3’,5,5’-tetramethylbenzidine. Plates were read in an ELISA plate reader at 450 nm after acidification. The intensity of the color reaction is directly proportional to the concentration of hTL1A in the samples [Manual TL1A, Soluble (Human) Detection Set for ELISA Application, Apotech, Epalinges, Switzerland]. Statistical Analysis Statistical analyses were carried out using SPSS 11.5 and STATISTICA 6.0. Since the data were distributed nonnormally, the Kruskal–Wallis test, the Mann–Whitney U test, and Spearman’s correlation analysis were used in comparisons between groups. A value of p<0.05 was considered to represent a statistically significant result. […]

Pipeline specifications

Software tools SPSS, Statistica
Application Miscellaneous
Organisms Homo sapiens
Diseases Anemia, Sickle Cell, Hypertension, Pulmonary, Neoplasms, Acute Chest Syndrome