48孔板荧光素酶报告基因实验边缘效应
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摘要
为了研究48孔板荧光素酶报告基因实验边缘效应以及寻找可能消除边缘效应的方法措施。本研究用常规方法培养人胚肾细胞HEK293,运用脂质体转染试剂Lipofectamin 2000将荧光素酶报告基因质粒p GL4.13Z和内参质粒p RL-TK共转染入48孔板中的HEK 293细胞。转染后24 h用荧光素酶报告基因实验检测试剂测量细胞的荧光素酶活性,比较角落孔、边缘孔以及中间孔测定值的差异,以及内参照海肾荧光素酶活性校准后的差值,并通过细胞计数来观察此种差异是否与细胞数目和转染效率相关。在此基础上,寻找可能减小边缘效应的方法,如细胞种植入48孔板后将其室温静置、48孔板重叠、放弃使用边缘孔。运用48孔细胞培养板进行荧光素酶报告基因实验存在边缘效应,边缘孔与中间孔的荧光素酶活性测定值有显著的差异且有统计学意义(p<0.05),而边缘效应的产生与细胞数目和转染效率无明显关联。细胞种植入48孔板后将其室温静置1.5 h、或重叠一个48孔板以及空置边缘孔都不能减弱边缘效应;然而将48孔板四周边缘孔填充液体(磷酸盐缓冲液(1*PBS),水),可以削弱边缘效应。用48孔细胞培养板进行荧光素酶报告基因实验会产生边缘效应,并可能造成对实验结果的误判;48孔板四周边缘孔填充相应的液体可以削弱边缘效应。
In order to investigate the edge effects of 48-well plates luciferase reporter gene assay and its elimination methods, we took the conventional methods to culture human embryo kidney cells(HEK293), and transfected the luciferase reporter gene plasmid p GL4.13 Z and internal reference plasmid p RL-TK with the transfection reagent Lipofectamine 2000 into HEK293 cells of 48-well plate. Luciferase activity was measured with the luciferase reporter gene experiment reagent 24 h after transfection. We compared the differences of the tested values among corner wells, edge wells and central wells, and the D-value of the activity of RLus after calibration. And cell counts were used to observe whether the difference was related to cell number and transfection efficiency. On this basis, we sought ways to reduce the edge effect like implanting the cells into the 48-well plates at the room temperature,overlapping the 48 plates, and abandoning the edge wells. Luciferase reporter gene assay cultured in 48-well plates had edge effects. There was significant difference in the determination of luciferase activity between the marginal and the middle wells(p<0.05). There was no significant correlation between the edge effects and cell numbers, and transfection efficiency. Pre-incubating plate with newly seeded cells at room temperature(RT), stacking one extra plate on the top of experimental plate, and avoiding use of the peripheral wells on plates were all useless for reducing edge effects; However, the edge effect could be weakened by filling the edge wells of 48-well plates with liquid(phosphate buffer(1*PBS) and water). The results of luciferase reporter gene assay performed with 48-well plates could be affected significantly by edge effects of 48-well plates, which might cause wrong interpretation of the result;and filling edge well with liquid could eliminate edge effects.
In order to investigate the edge effects of 48-well plates luciferase reporter gene assay and its elimination methods, we took the conventional methods to culture human embryo kidney cells(HEK293), and transfected the luciferase reporter gene plasmid p GL4.13 Z and internal reference plasmid p RL-TK with the transfection reagent Lipofectamine 2000 into HEK293 cells of 48-well plate. Luciferase activity was measured with the luciferase reporter gene experiment reagent 24 h after transfection. We compared the differences of the tested values among corner wells, edge wells and central wells, and the D-value of the activity of RLus after calibration. And cell counts were used to observe whether the difference was related to cell number and transfection efficiency. On this basis, we sought ways to reduce the edge effect like implanting the cells into the 48-well plates at the room temperature,overlapping the 48 plates, and abandoning the edge wells. Luciferase reporter gene assay cultured in 48-well plates had edge effects. There was significant difference in the determination of luciferase activity between the marginal and the middle wells(p<0.05). There was no significant correlation between the edge effects and cell numbers, and transfection efficiency. Pre-incubating plate with newly seeded cells at room temperature(RT), stacking one extra plate on the top of experimental plate, and avoiding use of the peripheral wells on plates were all useless for reducing edge effects; However, the edge effect could be weakened by filling the edge wells of 48-well plates with liquid(phosphate buffer(1*PBS) and water). The results of luciferase reporter gene assay performed with 48-well plates could be affected significantly by edge effects of 48-well plates, which might cause wrong interpretation of the result;and filling edge well with liquid could eliminate edge effects.
引文
Alam J.,and Cook J.L.,1990,Reporter genes:application to the study of mammalian gene transcription,Analytical Biochemistry,188(2):245-254
Carralot J.P.,Ogier A.,Boese A.,Genovesio A.,Brodin P.,Sommer P.,and Dorval T.,2012,A novel specific edge effect correction method for rna interference screenings,Bioinformatics,28(2):261-268
Choy G.,Choyke P.,and Libutti S.K.,2003,Current advances in molecular imaging:noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research,Molecular Imaging,2(4):303-312
de Wet J.R.,Wood K.V.,Helinski D.R.,and Deluca M.,1985,Cloning of firefly luciferase cdna and the expression of active luciferase in Escherichia coli,Proc.Nati.Acad.Sci.U.S.A.,82(23):7870-7873
Edinger M.,Cao Y.A.,Verneris M.R.,Bachmann M.H.,Contag C.H.,and Negrin R.S.,2003,Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging,Blood,101(2):640-648
Faessel H.M.,Levasseur L.M.,Slocum H.K.,and Greco W.R.,1999,Parabolic growth patterns in 96-well plate cell growth experiments,In Vitro Cellular and Developmental Biology-Animal,35(5):270-278
Gambhir S.S.,Barrio J.R.,Herschman H.R.,and Phelps M.E.,1999,Assays for noninvasive imaging of reporter gene expression,Nuclear Medicine and Biology,26(5):481-490
Golzio M.,Rols M.P.,Gabriel B.,and TeissiéJ.,2004,Optical imaging of in vivo gene expression:a critical assessment of the methodology and associated technologies,Gene Therapy,11(1):S85-S91
Gould S.J.,and Subramani S.,1988,Firefly luciferase as a tool in molecular and cell biology,Analytical Biochemistry,175(1):5-13
Ibrahim N.M.,Marinovic A.C.,Price S.R.,Young L.G.,and Fr觟hlich O.,2000,Pitfall of an internal control plasmid:response of renilla luciferase(p RL-TK)plasmid to dihydrotestosterone and dexamethasone,Biotechniques,29(4):782-784
Ignowski J.M.,and Schaffer D.V.,2004,Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells,Biotechnol.Bioeng.,86(7):827-834
Kricka L.J.,Carter T.J.,Burt S.M.,Kennedy J.H.,Holder R.L.,Halliday M.I.,Telford M.E.,and Wisdom G.B.,1980,Variability in the adsorption properties of microtitre plates used as solid supports in enzyme immunoassay,Clinical Chemistry,26(6):741-744
Lundholt B.K.,Scudder K.M.,and Pagliaro L.,2003,A simple technique for reducing edge effect in cell-based assays,J.Biomol.Screen,8(5):566-570
Naylor L.H.,1999,Reporter gene technology:the future looks bright,Biochem.Pharmacol.,58(5):749-757
Patel M.I.,Tuckerman R.,and Dong Q.,2005,A pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2h-tetrazolium(mts)assay due to evaporation in wells on the edge of a 96 well plate,Biotechnology Letters,27(11):805-808
Rice B.W.,Cable M.D.,and Nelson M.B.,2001,In vivo imaging of light-emitting probes,J.Biomed.Opt.,6(4):432-440
Rosenthal N.,1987,Identification of regulatory elements of cloned genes with functional assays,Methods.Enzymol.,152(1):704-720
Shekarchi I.C.,Sever J.L.,Lee Y.J.,Castellano G.,and Madden D.L.,1984,Evaluation of various plastic microtiter plates with measles,toxoplasma,and gamma globulin antigens in enzyme-linked immunosorbent assays,J.Clin.Microbiol.,19(2):89-96
Carralot J.P.,Ogier A.,Boese A.,Genovesio A.,Brodin P.,Sommer P.,and Dorval T.,2012,A novel specific edge effect correction method for rna interference screenings,Bioinformatics,28(2):261-268
Choy G.,Choyke P.,and Libutti S.K.,2003,Current advances in molecular imaging:noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research,Molecular Imaging,2(4):303-312
de Wet J.R.,Wood K.V.,Helinski D.R.,and Deluca M.,1985,Cloning of firefly luciferase cdna and the expression of active luciferase in Escherichia coli,Proc.Nati.Acad.Sci.U.S.A.,82(23):7870-7873
Edinger M.,Cao Y.A.,Verneris M.R.,Bachmann M.H.,Contag C.H.,and Negrin R.S.,2003,Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging,Blood,101(2):640-648
Faessel H.M.,Levasseur L.M.,Slocum H.K.,and Greco W.R.,1999,Parabolic growth patterns in 96-well plate cell growth experiments,In Vitro Cellular and Developmental Biology-Animal,35(5):270-278
Gambhir S.S.,Barrio J.R.,Herschman H.R.,and Phelps M.E.,1999,Assays for noninvasive imaging of reporter gene expression,Nuclear Medicine and Biology,26(5):481-490
Golzio M.,Rols M.P.,Gabriel B.,and TeissiéJ.,2004,Optical imaging of in vivo gene expression:a critical assessment of the methodology and associated technologies,Gene Therapy,11(1):S85-S91
Gould S.J.,and Subramani S.,1988,Firefly luciferase as a tool in molecular and cell biology,Analytical Biochemistry,175(1):5-13
Ibrahim N.M.,Marinovic A.C.,Price S.R.,Young L.G.,and Fr觟hlich O.,2000,Pitfall of an internal control plasmid:response of renilla luciferase(p RL-TK)plasmid to dihydrotestosterone and dexamethasone,Biotechniques,29(4):782-784
Ignowski J.M.,and Schaffer D.V.,2004,Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells,Biotechnol.Bioeng.,86(7):827-834
Kricka L.J.,Carter T.J.,Burt S.M.,Kennedy J.H.,Holder R.L.,Halliday M.I.,Telford M.E.,and Wisdom G.B.,1980,Variability in the adsorption properties of microtitre plates used as solid supports in enzyme immunoassay,Clinical Chemistry,26(6):741-744
Lundholt B.K.,Scudder K.M.,and Pagliaro L.,2003,A simple technique for reducing edge effect in cell-based assays,J.Biomol.Screen,8(5):566-570
Naylor L.H.,1999,Reporter gene technology:the future looks bright,Biochem.Pharmacol.,58(5):749-757
Patel M.I.,Tuckerman R.,and Dong Q.,2005,A pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2h-tetrazolium(mts)assay due to evaporation in wells on the edge of a 96 well plate,Biotechnology Letters,27(11):805-808
Rice B.W.,Cable M.D.,and Nelson M.B.,2001,In vivo imaging of light-emitting probes,J.Biomed.Opt.,6(4):432-440
Rosenthal N.,1987,Identification of regulatory elements of cloned genes with functional assays,Methods.Enzymol.,152(1):704-720
Shekarchi I.C.,Sever J.L.,Lee Y.J.,Castellano G.,and Madden D.L.,1984,Evaluation of various plastic microtiter plates with measles,toxoplasma,and gamma globulin antigens in enzyme-linked immunosorbent assays,J.Clin.Microbiol.,19(2):89-96