Computational protocol: Association of innate defense proteins BPIFA1 and BPIFB1 with disease severity in COPD

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Protocol publication

[…] Lung tissue blocks were submersed in RNA later (Qiagen, Hilden, Germany) and stored at −80°C. RNA extraction was performed by using the miRNeasy Mini kit (Qiagen). Next, cDNA was prepared by using the iScript Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Taq-Man® Gene Expression Assays (Applied Biosystems, Forster City, CA, USA) were used to measure the expression of the target genes BPIFA1 and BPIFB1 and the reference genes glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase-1, and succinate dehydrogenase complex flavoprotein subunit A. qRT-PCRs were performed in duplicate, and a standard curve was created by serial dilutions of a mixture of all samples. The amplifications were performed by using a LightCycler 96 detection system (Roche, Basel, Switzerland). The amplification conditions consisted of a preincubation step (600 s at 95°C), followed by 50 cycles of a two-step amplification (10 s at 95°C and 15 s at 60°C). Data were analyzed using the standard curve method, and the expressions of BPIFA1 and BPIFB1 were calculated relative to the expression of the three reference genes, using the geNorm applet ( [...] Statistical analyses were performed by using Sigma Stat software (SPSS Statistics 23, Chicago, IL, USA) including Kruskal–Wallis, Mann–Whitney U-test, Fisher’s exact test, general linear model, and Spearman correlation analysis. Differences at p-values <0.05 were considered to be significant. Characteristics of the study population ( and ) are expressed as median and interquartile range. […]

Pipeline specifications

Software tools geNorm, SPSS
Databases MedGen
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Pulmonary Disease, Chronic Obstructive