Selection of Suitable Housekeeping Genes for Real-Time Quantitative PCR in CD4+ Lymphocytes from Asthmatics with or without Depression

Abstract

ObjectiveNo optimal housekeeping genes (HKGs) have been identified for CD4+ T cells from non-depressive asthmatic and depressive asthmatic adults for normalizing quantitative real-time PCR (qPCR) assays. The aim of present study was to select appropriate HKGs for gene expression analysis in purified CD4+ T cells from these asthmatics.MethodsThree groups of subjects (Non-depressive asthmatic, NDA, n = 10, Depressive asthmatic, DA, n = 11, and Healthy control, HC, n = 10 respectively) were studied. qPCR for 9 potential HKGs, namely RNA, 28S ribosomal 1 (RN28S1), ribosomal protein, large, P0 (RPLP0), actin, beta (ACTB), cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK1), beta-2-microglobulin (B2M), glucuronidase, beta (GUSB) and ribosomal protein L13a (RPL13A), was performed. Then the data were analyzed with three different applications namely BestKeeper, geNorm, and NormFinder.ResultsThe analysis of gene expression data identified B2M and RPLP0 as the most stable reference genes and showed that the level of PPIA was significantly different among subjects of three groups when the two best HKGs identified were applied. Post-hoc analysis by Student-Newman-Keuls correction shows that depressive asthmatics and non-depressive asthmatics exhibited lower expression level of PPIA than healthy controls (p<0.05).Conclusions B2M and RPLP0 were identified as the most optimal HKGs in gene expression studies involving human blood CD4+ T cells derived from normal, depressive asthmatics and non-depressive asthmatics. The suitability of using the PPIA gene as the HKG for such studies was questioned due to its low expression in asthmatics.

Selection and validation of reference house­keeping genes in the J774A1 macrophage cell line for quantitative real-time PCR

“…The selected set of candidate reference genes ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, PPIA, and RPL13A are associated with a wide variety of cellular functions (Nelissen et al, 2010;Wang et al, 2012). Therefore, the identification of reliable reference genes across various developmental and experimental conditions is required to avoid unstable or misleading qPCR results.…”

Th1/Th2/Th17 cells imbalance in patients with asthma with and without psychological symptoms

“…39 The expressions of T-bet, GATA-3, and RORC were normalized by housekeeping gene ␤-2-microglobulin (B2M). 40 Allergy and Asthma Proceedings 149 …”

Ficolin-2 Inhibits Hepatitis C Virus Infection, whereas Apolipoprotein E3 Mediates Viral Immune Escape

“…RNA was quantified using a real-time RT-PCR with the SYBR Green real-time PCR Master Mix kit (Toyobo). The primers P1 (59-GGCGACCTGGAAGTCCAACT-39) and P2 (59-CCATCAGCACCA CAGCCTTC-39) were used for the endogenous control gene ribosomal protein, large, P0 (31). G3PDH forward (59-ACCACAGTCCATGCCATC-AC-39) and G3PDH reverse (59-TCCACCACCCTGTTGCTGTA-39) were used for another housekeeping gene, G3PDH.…”