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三叶草王铁虫 (小有名气)
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[求助]
英译汉，谢谢
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1.Transfections with has-miR-29a, -29b-1, -29c mimics and negative control (Ambion, Austin, TX, USA) were performed at a concentration of 100 nM with Lipofectamine 2000 (Invitrogen,Carlsbad, CA, USA). The expressions of DNMT1, DNMT3A and DNMT3B were knocked down stably in A549 and H1299 cell lines。using a lentiviral shRNA system from Santa Cruz Biotechnology (Dallas, Texas, USA) with puromycin selection. Transient knock down of WIF-1 was achieved by using WIF-1 shRNA Plasmid (Santa Cruz) according to the manufacturer’s instructions. For the demethylation assay, 5-Aza-deoxycytidine (5-Aza, Sigma–Aldrich, St. Louis, MO, USA) was added at a concentration of 10lM for 72 h 2 . Methylation-Specific PCR (MSP) Genomic DNA was extracted with the DNA Maxi Kit (Qiagen, Valencia, CA), and modified with sodium bisulfite using the MethylCode Bisulfite Conversion Kit (Invitrogen) according to the manufacturer’s protocol. The methylation-specific or unmethylation-specific primer (MSP or USP) for WIF-1 was designed according to a previous report[7](Table 1). MiR-29s are transcribed into two primary transcripts from two chromosomes (miR-29b1and miR-29a at chromosome 7q32; miR-29b2 and miR-29c at chromosome 1q32). The MSP and USP were designed using the MethPrimer program (https://www.urogene.org/cgi-bin/methprimer/methprimer). Primers for miR-29b1 and miR-29a correspond to the promoter region sequences 1112 to1085 and993 to 971, respectively (Table 1). Primers for miR-29b2 and miR-29c correspond to the promoter region sequences 1339 to1314 and1178 to1153, respectively (Table 1). 3. Cell proliferation assays Cancer cells were transfected with microRNA mimics and/or WIF-1 shRNA. Seventy-two hours after transfection, cells wereseeded into 96-well plates (510 3 cells per well), and incubated at 37C. The number of viable cells was measured at daily intervals. A volume of 10lL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated for 4 h. The precipitate formed was dissolved by addition of 100lL of dimethyl sulfoxide and absorbance was measured at 562 nm using an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA). Each experiment was performed in triplicate and repeated three times 4. Apoptosis assay Cells were seeded in 6-well plates at a density of 110 6 cells per well and transfected with microRNA mimics and/or WIF-1 shRNA. Seventy-two hours after transfection, the cells were resuspended and stained with FITC-conjugated anti-annexin V antibody and propidium iodide (PI) using the Annexin V-FITC Apoptosis Detection Kit (Sigma–Aldrich). Stained cells were then quantified by FACSCalibur flow cytometry (Becton Dickinson, USA). 5. Statistical analysis Differences between two groups were assessed by the Student’s t-test or Mann–Whitney’s U-test. Correlations were assessed by Pearson’s correlation test. Variance analysis between multiple groups was performed by one-way ANOVA. The data were presented as mean ± standard deviation (SD) andp< 0.05 was considered statistically significant |
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至尊木虫 (知名作家)
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三叶草王: 金币+25, 翻译EPI+1, ★★★★★最佳答案 2014-08-28 09:57:43
三叶草王: 金币+25, 翻译EPI+1, ★★★★★最佳答案 2014-08-28 09:57:43
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刚才忘了删掉部分原文，以下文为准： 1. 采用Lipofectamine 2000 （Invitrogen,Carlsbad, CA, USA）转染hsa-miR-29a、 -29b-1、-29c（模拟物）以及阴性对照 (Ambion, Austin, TX, USA) ，浓度为100 nM。在A549和H1299细胞中利用慢病毒载体shRNA系统（含有嘌呤霉素筛选标记）（Santa Cruz Biotechnology，Dallas, Texas, USA）稳定转染方法敲减DNMT1、DNMT3A和DNMT3B的表达。根据生产商说明书利用WIF-1 shRNA Plasmid (Santa Cruz)瞬时敲减WIF-1（的表达）。去甲基化实验时，用10 μM 5-Aza （Sigma–Aldrich, St. Louis, MO, USA) 处理72小时。 2 . 甲基化特异性PCR (MSP) 用DNA Maxi Kit(Qiagen, Valencia, CA)提取基因组DNA，并用MethylCode™ Bisulfite Conversion Kit （Invitrogen）根据生产商使用手册进行亚硫酸氢钠进行修饰。根据以前的报告[7]设计WIF-1甲基化特异性引物和非甲基化特异引物（MSP或USP）(表1)。MiR-29s从两个不同染色体上转录成两个主要转录产物（miR-29b1和miR-29a在染色体7q32; miR-29b2和miR-29c在染色体1q32）。MSP和USP用MethPrimer程序设计 (https://www.urogene.org/cgi-bin/methprimer/methprimer)。miR-29b1引物和miR-29a引物分别对应于启动子区域顺序1112到1085、及993到971 (表1)。miR-29b2引物和miR-29c引物分别对应于启动子顺序的1339至1314和1178至1153(表1)。 3. 细胞增殖实验 肿瘤细胞转染microRNA模拟物和/或WIF-1 shRNA。转染后72小时，细胞种植于96孔板（5000细胞/孔），并于37度培养。每天计数活细胞数量。每孔加入10 μl 5 mg/ml的MTT并继续培养4小时。所形成的沉淀溶于100 μl而家亚砜，并用ELISA酶标仪 (Bio-Rad Laboratories, Hercules, CA, USA)检测562 nm的光吸收度。每一实验设三个平行实验，同样的实验重复三次。 4. 凋亡检测 细胞种植于6孔板中，密度为每孔1百万细胞，用microRNA模拟物和/或WIF-1 shRNA转染。转染后72小时，再次悬浮细胞并用FITC交联的抗膜联蛋白V抗体和碘化丙啶（PI）对细胞染色，染色采用膜联蛋白V-FITC凋亡检测试剂盒 (Sigma–Aldrich)进行。用FACSCalibur流式细胞仪 (Becton Dickinson, USA)对染色细胞进行定量。 5. 用Student’s t检验或Mann–Whitney’s U检验对两组间的差异进行统计学分析。用Pearson’s的相关检验进行相关分析。通过单因素方差分析进行多组间方差分析。数据用均数±标准差(SD)表示，视p< 0.05为有显著统计学意义。 |
3楼2014-08-28 08:56:52
ssssllllnnnn
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1. 采用Lipofectamine 2000 （Invitrogen,Carlsbad, CA, USA）转染hsa-miR-29a、 -29b-1、-29c（模拟物）以及阴性对照 (Ambion, Austin, TX, USA) ，浓度为100 nM。在A549和H1299细胞中利用慢病毒载体shRNA系统（含有嘌呤霉素筛选标记）（Santa Cruz Biotechnology，Dallas, Texas, USA）稳定转染方法敲减DNMT1、DNMT3A和DNMT3B的表达。根据生产商说明书利用WIF-1 shRNA Plasmid (Santa Cruz)瞬时敲减WIF-1（的表达）。去甲基化实验时，用10 μM 5-Aza （Sigma–Aldrich, St. Louis, MO, USA) 处理72小时。 2 . 甲基化特异性PCR (MSP) 用DNA Maxi Kit(Qiagen, Valencia, CA)提取基因组DNA，并用MethylCode™ Bisulfite Conversion Kit （Invitrogen）根据生产商使用手册进行亚硫酸氢钠进行修饰。根据以前的报告[7]设计WIF-1甲基化特异性引物和非甲基化特异引物（MSP或USP）(表1)。MiR-29s从两个不同染色体上转录成两个主要转录产物（miR-29b1和miR-29a在染色体7q32; miR-29b2和miR-29c在染色体1q32）。MSP和USP用MethPrimer程序设计 (https://www.urogene.org/cgi-bin/methprimer/methprimer)。 The MSP and USP were designed using the MethPrimer program (https://www.urogene.org/cgi-bin/methprimer/methprimer)。miR-29b1引物和miR-29a引物分别对应于启动子区域顺序1112到1085、及993到971 (表1)。miR-29b2引物和miR-29c引物分别对应于启动子顺序的1339至1314和1178至1153(表1)。 3. 细胞增殖实验 肿瘤细胞转染microRNA模拟物和/或WIF-1 shRNA。转染后72小时，细胞种植于96孔板（5000细胞/孔），并于37度培养。每天计数活细胞数量。每孔加入10 μl 5 mg/ml的MTT并继续培养4小时。所形成的沉淀溶于100 μl而家亚砜，并用ELISA酶标仪 (Bio-Rad Laboratories, Hercules, CA, USA)检测562 nm的光吸收度。每一实验设三个平行实验，同样的实验重复三次。 4. 凋亡检测 细胞种植于6孔板中，密度为每孔1百万细胞，用microRNA模拟物和/或WIF-1 shRNA转染。转染后72小时，再次悬浮细胞并用FITC交联的抗膜联蛋白V抗体和碘化丙啶（PI）对细胞染色，染色采用膜联蛋白V-FITC凋亡检测试剂盒 (Sigma–Aldrich)进行。用FACSCalibur流式细胞仪 (Becton Dickinson, USA)对染色细胞进行定量。 5. 用Student’s t检验或Mann–Whitney’s U检验对两组间的差异进行统计学分析。用Pearson’s的相关检验进行相关分析。通过单因素方差分析进行多组间方差分析。数据用均数±标准差(SD)表示，视p< 0.05为有显著统计学意义。 |
2楼2014-08-28 08:53:51