Selection of Suitable Housekeeping Genes for Real-Time Quantitative PCR in CD4+ Lymphocytes from Asthmatics with or without Depression
نویسندگان
چکیده
OBJECTIVE No 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. METHODS Three 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. RESULTS The 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.
منابع مشابه
Selection of suitable reference genes for real-time PCR studies of early developmental stages of sturgeons
In quantitative real-time PCR, the mRNA level can be quantified in relative terms based on the expression ratio of mRNAs of the target gene and an internal reference gene. Since, an internal standard should be expressed at a constant level among different tissues of an organism at all stages of development, and should be unaffected by the experimental treatment, the stability of different refer...
متن کاملSelection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells.
The stability of housekeeping genes (HKGs) is critical when performing real-time quantitative PCR. To date, the stability of common HKGs has not been systematically compared in human airway epithelial cells (AEC) in normal and atopic subjects. Expression levels of 12 HKGs were measured in AECs from a cohort of 30 healthy atopic nonasthmatic or atopic asthmatic children. Gene expression stabilit...
متن کاملValidation of Reference Genes for Real Time PCR Normalization in Milk Somatic Cells of Holstein Dairy Cattle
Real time-qPCR is the most reliable method for evaluation of mRNA expression levels. However, to obtain accurate results, selection of suitable reference genes is necessary for normalizing the real-time qPCR data. The aim of this research was to validate the expression stability of three potential reference genes (ACTB, GAPDH and UXT) in milk somatic cells of Holstein dairy cattle under differe...
متن کاملQuantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.
Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be sui...
متن کاملO-35: Over-Expression of XRCC1 As Potential Biomarker for Poor Prognosis in Human Preimplantation Embryos: Selection by Study of 84 Genes Involved in DNA Damage Signaling Pathways
Background: Chromosome abnormalities are associated with poor morphology and development in human preimplantation embryos, all together lead to poor outcomes. This study aimed to explore altered expression of DNA damage pathways in “poor morphological and development embryos with sever aneuploidies”. Materials and Methods: Surplus day-4 embryos of PGD cases were pooled in two groups: Poor progn...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
دوره 7 شماره
صفحات -
تاریخ انتشار 2012