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 (http://medgen.ugent.be/~jvdesomp/genorm/). [...] 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