Is a useful technique for acquiring the gene expression pattern of a number of selected genes due to its high sensitivity, specificity and broad quantification range [8]. To obtain accurate and reliable gene expression quantification, normalization of gene expression data against housekeeping genes (HKGs) is particularly important. For this purpose, an ideal HKG should be either stably expressed across experimental conditions or similarly expressed among samples affected by different disease processes. However, commonly used HKGs vary considerably in different disease processes or different tissue and cell types. Thus, it is important to performSelection of Suitable Housekeeping GenesFigure 1. Flow cytometry analysis on CD4+ T cells 18334597 in lymphocyte suspension and in purified CD4+ cells by immunomagnetic depletion with the human CD4+ T Cell Isolation Kit II. Cells were directly stained with conjugated fluorescently labeled antibodies for CD4 (BD Biosciences) in lymphocyte suspension and in purified CD4+ samples. doi:10.1371/journal.pone.0048367.grigorous validation of the most stable HKGs in different tissues or cells and/or disease status before commencement of any qPCR study. Before comparison of gene expression profiling of CD4+ T cells in pure asthmatics or depressive asthmatics, this requirement must be met. However, there have been no studies that have systematically compared the stability of common HKGs in such conditions. In the present study, we carried out a careful evaluation of 9 HKGs in uncultured human CD4+ T cells derived from healthy individuals, non-depressive asthmatics and depressive asthmatics. After analysis and comparison using three different statistical methods, B2M and RPLP0 were identified as the most suitable HKGs for gene expression studies in uncultured CD4+ T cells of asthmatics with or without depression.(HRSD) [9], which is observer-rated and a score 8 was used as a cut-off point for comorbid depressive symptoms. The 1676428 clinical control of Erastin web asthma was assessed with Asthma Control Test (ACT) [10] and health-related quality of life was appraised with the Standardized Version of the Asthma Quality of Life Questionnaire (AQLQs) [11]. HC subjects (n = 10) with no significant medical conditions were recruited by advertisement. Subjects were excluded if they had been taking oral glucocorticosteroids within 4 weeks of the blood draw. Other exclusion criteria included Epothilone D uncontrolled asthma or change in maintenance therapy, acute respiratory tract infection within 4 weeks, and current smoking. The present study received approval from the West China Hospital Institutional Review Board and all participants gave written informed consent.Materials and Methods PatientsThree groups of subjects were studied: asthmatics with depression (Depressive asthmatics, DA), asthmatics without depression, (Non-depressive asthmatics, NDA) and Healthy controls (HC). Patients of DA group (n = 11) and NDA group (n = 10) were enrolled from the outpatient clinic of the West China Hospital of Sichuan University from September 2011 to January 2012 as a cross-sectional study. All patients had symptoms consistent with diagnosis of asthma and demonstrated evidence of bronchodilator reversibility of .12 and 200 mL in forced expiratory volume in 1 s (FEV1) following 400 mg of inhaled salbutamol or provocative dose of methacholine causing a 20 drop in FEV1 (PD20FEV1) ,2.5 mg. A bronchial challenge test was performed for all of the patients, except one.Is a useful technique for acquiring the gene expression pattern of a number of selected genes due to its high sensitivity, specificity and broad quantification range [8]. To obtain accurate and reliable gene expression quantification, normalization of gene expression data against housekeeping genes (HKGs) is particularly important. For this purpose, an ideal HKG should be either stably expressed across experimental conditions or similarly expressed among samples affected by different disease processes. However, commonly used HKGs vary considerably in different disease processes or different tissue and cell types. Thus, it is important to performSelection of Suitable Housekeeping GenesFigure 1. Flow cytometry analysis on CD4+ T cells 18334597 in lymphocyte suspension and in purified CD4+ cells by immunomagnetic depletion with the human CD4+ T Cell Isolation Kit II. Cells were directly stained with conjugated fluorescently labeled antibodies for CD4 (BD Biosciences) in lymphocyte suspension and in purified CD4+ samples. doi:10.1371/journal.pone.0048367.grigorous validation of the most stable HKGs in different tissues or cells and/or disease status before commencement of any qPCR study. Before comparison of gene expression profiling of CD4+ T cells in pure asthmatics or depressive asthmatics, this requirement must be met. However, there have been no studies that have systematically compared the stability of common HKGs in such conditions. In the present study, we carried out a careful evaluation of 9 HKGs in uncultured human CD4+ T cells derived from healthy individuals, non-depressive asthmatics and depressive asthmatics. After analysis and comparison using three different statistical methods, B2M and RPLP0 were identified as the most suitable HKGs for gene expression studies in uncultured CD4+ T cells of asthmatics with or without depression.(HRSD) [9], which is observer-rated and a score 8 was used as a cut-off point for comorbid depressive symptoms. The 1676428 clinical control of asthma was assessed with Asthma Control Test (ACT) [10] and health-related quality of life was appraised with the Standardized Version of the Asthma Quality of Life Questionnaire (AQLQs) [11]. HC subjects (n = 10) with no significant medical conditions were recruited by advertisement. Subjects were excluded if they had been taking oral glucocorticosteroids within 4 weeks of the blood draw. Other exclusion criteria included uncontrolled asthma or change in maintenance therapy, acute respiratory tract infection within 4 weeks, and current smoking. The present study received approval from the West China Hospital Institutional Review Board and all participants gave written informed consent.Materials and Methods PatientsThree groups of subjects were studied: asthmatics with depression (Depressive asthmatics, DA), asthmatics without depression, (Non-depressive asthmatics, NDA) and Healthy controls (HC). Patients of DA group (n = 11) and NDA group (n = 10) were enrolled from the outpatient clinic of the West China Hospital of Sichuan University from September 2011 to January 2012 as a cross-sectional study. All patients had symptoms consistent with diagnosis of asthma and demonstrated evidence of bronchodilator reversibility of .12 and 200 mL in forced expiratory volume in 1 s (FEV1) following 400 mg of inhaled salbutamol or provocative dose of methacholine causing a 20 drop in FEV1 (PD20FEV1) ,2.5 mg. A bronchial challenge test was performed for all of the patients, except one.
