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三葉草王鐵蟲 (正式寫手)
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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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ssssllllnnnn 
至尊木蟲 (知名作家)
Translator and Proofreader
- 翻譯EPI: 1690
- 應(yīng)助: 452 (碩士)
- 金幣: 31580.9
- 紅花: 100
- 帖子: 7681
- 在線: 19966.6小時
- 蟲號: 3328089
- 注冊: 2014-07-17
- 專業(yè): 腫瘤發(fā)生
【答案】應(yīng)助回帖
|
1. 采用Lipofectamine 2000 （Invitrogen,Carlsbad, CA, USA）轉(zhuǎn)染hsa-miR-29a、 -29b-1、-29c（模擬物）以及陰性對照 (Ambion, Austin, TX, USA) ，濃度為100 nM。在A549和H1299細胞中利用慢病毒載體shRNA系統(tǒng)（含有嘌呤霉素篩選標記）（Santa Cruz Biotechnology，Dallas, Texas, USA）穩(wěn)定轉(zhuǎn)染方法敲減DNMT1、DNMT3A和DNMT3B的表達。根據(jù)生產(chǎn)商說明書利用WIF-1 shRNA Plasmid (Santa Cruz)瞬時敲減WIF-1（的表達）。去甲基化實驗時，用10 μM 5-Aza （Sigma–Aldrich, St. Louis, MO, USA) 處理72小時。 2 . 甲基化特異性PCR (MSP) 用DNA Maxi Kit(Qiagen, Valencia, CA)提取基因組DNA，并用MethylCode™ Bisulfite Conversion Kit （Invitrogen）根據(jù)生產(chǎn)商使用手冊進行亞硫酸氫鈉進行修飾。根據(jù)以前的報告[7]設(shè)計WIF-1甲基化特異性引物和非甲基化特異引物（MSP或USP）(表1)。MiR-29s從兩個不同染色體上轉(zhuǎn)錄成兩個主要轉(zhuǎn)錄產(chǎn)物（miR-29b1和miR-29a在染色體7q32; miR-29b2和miR-29c在染色體1q32）。MSP和USP用MethPrimer程序設(shè)計 (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引物分別對應(yīng)于啟動子區(qū)域順序1112到1085、及993到971 (表1)。miR-29b2引物和miR-29c引物分別對應(yīng)于啟動子順序的1339至1314和1178至1153(表1)。 3. 細胞增殖實驗 腫瘤細胞轉(zhuǎn)染microRNA模擬物和/或WIF-1 shRNA。轉(zhuǎn)染后72小時，細胞種植于96孔板（5000細胞/孔），并于37度培養(yǎng)。每天計數(shù)活細胞數(shù)量。每孔加入10 μl 5 mg/ml的MTT并繼續(xù)培養(yǎng)4小時。所形成的沉淀溶于100 μl而家亞砜，并用ELISA酶標儀 (Bio-Rad Laboratories, Hercules, CA, USA)檢測562 nm的光吸收度。每一實驗設(shè)三個平行實驗，同樣的實驗重復(fù)三次。 4. 凋亡檢測 細胞種植于6孔板中，密度為每孔1百萬細胞，用microRNA模擬物和/或WIF-1 shRNA轉(zhuǎn)染。轉(zhuǎn)染后72小時，再次懸浮細胞并用FITC交聯(lián)的抗膜聯(lián)蛋白V抗體和碘化丙啶（PI）對細胞染色，染色采用膜聯(lián)蛋白V-FITC凋亡檢測試劑盒 (Sigma–Aldrich)進行。用FACSCalibur流式細胞儀 (Becton Dickinson, USA)對染色細胞進行定量。 5. 用Student’s t檢驗或Mann–Whitney’s U檢驗對兩組間的差異進行統(tǒng)計學(xué)分析。用Pearson’s的相關(guān)檢驗進行相關(guān)分析。通過單因素方差分析進行多組間方差分析。數(shù)據(jù)用均數(shù)±標準差(SD)表示，視p< 0.05為有顯著統(tǒng)計學(xué)意義。 |
2樓2014-08-28 08:53:51
ssssllllnnnn
至尊木蟲 (知名作家)
Translator and Proofreader
- 翻譯EPI: 1690
- 應(yīng)助: 452 (碩士)
- 金幣: 31580.9
- 紅花: 100
- 帖子: 7681
- 在線: 19966.6小時
- 蟲號: 3328089
- 注冊: 2014-07-17
- 專業(yè): 腫瘤發(fā)生
【答案】應(yīng)助回帖
★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★
三葉草王: 金幣+25, 翻譯EPI+1, ★★★★★最佳答案 2014-08-28 09:57:43
三葉草王: 金幣+25, 翻譯EPI+1, ★★★★★最佳答案 2014-08-28 09:57:43
|
剛才忘了刪掉部分原文，以下文為準： 1. 采用Lipofectamine 2000 （Invitrogen,Carlsbad, CA, USA）轉(zhuǎn)染hsa-miR-29a、 -29b-1、-29c（模擬物）以及陰性對照 (Ambion, Austin, TX, USA) ，濃度為100 nM。在A549和H1299細胞中利用慢病毒載體shRNA系統(tǒng)（含有嘌呤霉素篩選標記）（Santa Cruz Biotechnology，Dallas, Texas, USA）穩(wěn)定轉(zhuǎn)染方法敲減DNMT1、DNMT3A和DNMT3B的表達。根據(jù)生產(chǎn)商說明書利用WIF-1 shRNA Plasmid (Santa Cruz)瞬時敲減WIF-1（的表達）。去甲基化實驗時，用10 μM 5-Aza （Sigma–Aldrich, St. Louis, MO, USA) 處理72小時。 2 . 甲基化特異性PCR (MSP) 用DNA Maxi Kit(Qiagen, Valencia, CA)提取基因組DNA，并用MethylCode™ Bisulfite Conversion Kit （Invitrogen）根據(jù)生產(chǎn)商使用手冊進行亞硫酸氫鈉進行修飾。根據(jù)以前的報告[7]設(shè)計WIF-1甲基化特異性引物和非甲基化特異引物（MSP或USP）(表1)。MiR-29s從兩個不同染色體上轉(zhuǎn)錄成兩個主要轉(zhuǎn)錄產(chǎn)物（miR-29b1和miR-29a在染色體7q32; miR-29b2和miR-29c在染色體1q32）。MSP和USP用MethPrimer程序設(shè)計 (https://www.urogene.org/cgi-bin/methprimer/methprimer)。miR-29b1引物和miR-29a引物分別對應(yīng)于啟動子區(qū)域順序1112到1085、及993到971 (表1)。miR-29b2引物和miR-29c引物分別對應(yīng)于啟動子順序的1339至1314和1178至1153(表1)。 3. 細胞增殖實驗 腫瘤細胞轉(zhuǎn)染microRNA模擬物和/或WIF-1 shRNA。轉(zhuǎn)染后72小時，細胞種植于96孔板（5000細胞/孔），并于37度培養(yǎng)。每天計數(shù)活細胞數(shù)量。每孔加入10 μl 5 mg/ml的MTT并繼續(xù)培養(yǎng)4小時。所形成的沉淀溶于100 μl而家亞砜，并用ELISA酶標儀 (Bio-Rad Laboratories, Hercules, CA, USA)檢測562 nm的光吸收度。每一實驗設(shè)三個平行實驗，同樣的實驗重復(fù)三次。 4. 凋亡檢測 細胞種植于6孔板中，密度為每孔1百萬細胞，用microRNA模擬物和/或WIF-1 shRNA轉(zhuǎn)染。轉(zhuǎn)染后72小時，再次懸浮細胞并用FITC交聯(lián)的抗膜聯(lián)蛋白V抗體和碘化丙啶（PI）對細胞染色，染色采用膜聯(lián)蛋白V-FITC凋亡檢測試劑盒 (Sigma–Aldrich)進行。用FACSCalibur流式細胞儀 (Becton Dickinson, USA)對染色細胞進行定量。 5. 用Student’s t檢驗或Mann–Whitney’s U檢驗對兩組間的差異進行統(tǒng)計學(xué)分析。用Pearson’s的相關(guān)檢驗進行相關(guān)分析。通過單因素方差分析進行多組間方差分析。數(shù)據(jù)用均數(shù)±標準差(SD)表示，視p< 0.05為有顯著統(tǒng)計學(xué)意義。 |
3樓2014-08-28 08:56:52
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