Computational protocol: Urinary beta-trace protein gene expression analysis in type 2 diabetes mellitus patients

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Protocol publication

[…] Patients diagnosed with type 2 diabetes mellitus (T2DM) with a history of at least 5 years of disease determined in accordance with the criteria established by the American Diabetes Association were recruited between 2013 and 2014 among nephrology and endocrinology outpatients in the metropolitan area of São Paulo. The exclusion criteria included the existence of infection, an active malignant neoplasm at the moment of enrolment, presence of acquired immunodeficiency syndrome, and a diagnosis of end-stage renal disease, under conservative treatment or dialysis.This cross-sectional study was designed to evaluate the expression of BTP during diabetes, and did not involve changes in the treatment regimen of the participants. The two groups studied were classified by the presence or absence of T2DM.The inclusion criteria for the Control Group were absence of diagnosis of diabetes and chronic kidney disease, and no family history of diabetes and chronic kidney disease.Samples of blood and urine were collected at the time of recruitment to analyze levels of fasting blood glucose, glycated hemoglobin, serum creatinine and urinary BTP.Urinary BTP gene expression was measured by isolating urinary RNA (1μg, initially), which was then converted into first-strand cDNA with the aid of SSIII First Strand qPCR Supermix (Invitrogen, catalog number 11,752,050), according to the manufacturer's protocol.The expression of the specific BTP gene was assessed by quantitative real-time polymerase chain reaction (RT-qPCR). To standardize and determine the relative expression of the target gene, the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also expressed. The specific primers of the target and reference genes were designed with the help of the software Primer3 Input 0.4.0 and were checked for their specificity by the Primer-BLAST program.For RT-qPCR, an Applied Biosystems 7500 Real Time PCR Systems (Applied Biosystems, Foster City, USA) thermal cycler was used. Each sample had a final volume of 15μL, containing 1 x SYBR Green mix (Quantitec SYBR Green PCR kit, QIAGEN Catalog no. 204 054), 10pmol of each specific primer and 2μL of 10x-diluted cDNA. The parameters of amplification included an initial hot start at 95°C for 15 seconds, and 60°C for the primer sequence.To verify differences in urinary beta-trace protein gene expression between groups, the mean and standard deviation were analyzed using the Student's t test for independent samples. The median and first and third quartiles were analyzed using the Mann-Whitney test.To verify the expression of urinary BTP gene above and below the cutoff point of 6.34, as established in the literature, the relative frequency was used. The verification of a possible association between the quantities expressed between the two groups was performed using the χ2 test.To determine the sensitivity and specificity of BTP as a predictor of diabetes, a receiver operating characteristic (ROC) curve was used. The chance of positive or negative results in diabetic patients based upon the expression of BTP was evaluated, respectively, by positive and negative likelihood ratios. The significance level was 95%. Statistical analyses were performed using Stata software version 11.0® (USA).This study was approved by the Research Ethics Committee of the Faculdade de Medicina do ABC, under number 295.177, CAAE: 16235413.0.0000.0082. […]

Pipeline specifications

Software tools Primer3, Primer-BLAST
Application qPCR
Organisms Homo sapiens
Diseases Diabetes Mellitus
Chemicals Glucose