PD‐1 mRNA expression in peripheral blood mononuclear cells as a biomarker for different stages of primary gouty arthritis

There is currently a lack of biomarkers to assist the diagnosis and prediction of primary gouty arthritis (PG). Therefore, we evaluated the clinical value of programmed cell death protein 1 (PD‐1) mRNA expression in peripheral blood mononuclear cells (PBMCs) of patients with PG. This study included 36 patients with acute phase PG (APPG), 48 with non‐acute phase PG (NAPPG), 42 with asymptomatic hyperuricemia (AH) and 79 normal controls (NCs). PD‐1 mRNA expression levels were detected by qRT‐PCR. PD‐1 mRNA expression was statistically analysed by ANOVA or t tests, while correlations between PD‐1 mRNA and clinical variables were assessed using Pearson correlation tests. Receiver operator characteristic (ROC) curve analysis was used to evaluate the diagnostic value of PD‐1 in different PG stages. PD‐1 mRNA expression was significantly lower in patients with APPG than that in NAPPG, AH and NCs (P < 0.01). Correlation analysis revealed that PD‐1 mRNA levels correlated negatively with T‐score (r = ?0.209, P < 0.01). ROC curve analysis showed that serum uric acid (SUA), PD‐1 mRNA and both combined displayed higher diagnostic value in patients with PG, NAPPG and APPG compared to that in NCs and patients with non‐PG arthritis (NPG). Moreover, ROC curve analysis showed that SUA and PD‐1 mRNA had good diagnostic value in APPG, with the greatest diagnostic power when combined. PD‐1 mRNA could be a clinical auxiliary diagnostic biomarker for APPG, and the combined use of PD‐1 mRNA and SUA is better than that of SUA alone.     

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis : Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.