Selection of Suitable Housekeeping Genes for Real-Time Quantitative PCR in CD4+ Lymphocytes from Asthmatics with or without Depression 英文参考文献.docVIP

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Selection of Suitable Housekeeping Genes for Real-Time Quantitative PCR in CD4+ Lymphocytes from Asthmatics with or without Depression 英文参考文献.doc

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SelectionofSuitableHousekeepingGenesforReal-TimeQuantitativePCRinCD4LymphocytesfromAsthmaticswithorwithoutDepression英文参考文献

SelectionofSuitableHousekeepingGenesforReal-Time QuantitativePCRinCD4 LymphocytesfromAsthmatics withorwithoutDepression + TingWang1,Zong-AnLiang1,AndrewJ.Sandford2,Xing-YuXiong1,Yin-YinYang1,Yu-LinJi1* ,Jian- QingHe1* 1Department of Respiratory Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan Province, People’s Republic of China, 2The UBC James Hogg ResearchCentre,InstituteforHeart+LungHealth,St.Paul’sHospital,UniversityofBritishColumbia,Vancouver,BritishColumbia,Canada Abstract Objective:Nooptimalhousekeepinggenes(HKGs)havebeenidentifiedforCD4+Tcellsfromnon-depressiveasthmaticand depressiveasthmaticadultsfornormalizingquantitativereal-timePCR(qPCR)assays.Theaimofpresentstudywastoselect appropriateHKGsforgeneexpressionanalysisinpurifiedCD4 Tcellsfromtheseasthmatics. + Methods:Threegroupsofsubjects(Non-depressiveasthmatic,NDA,n=10,Depressiveasthmatic,DA,n=11,andHealthy control,HC,n=10respectively)werestudied.qPCRfor9potentialHKGs,namelyRNA,28Sribosomal1(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),wasperformed.ThenthedatawereanalyzedwiththreedifferentapplicationsnamelyBestKeeper,geNorm,and NormFinder. Results:TheanalysisofgeneexpressiondataidentifiedB2MandRPLP0asthemoststablereferencegenesandshowedthat 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 asthmaticsexhibitedlowerexpressionlevelofPPIAthanhealthycontrols(p,0.05). Conclusions:B2MandRPLP0wereidentifiedasthemostoptimalHKGsingeneexpressionstudiesinvolvinghumanblood CD4 Tcellsderivedfromnormal,depressiveasthmaticsandnon-depressiveasthmatics.ThesuitabilityofusingthePPIA geneastheHKGforsuchst