07/06 Lab Meeting- 小夏

1. The long-term kinetics (0-6 days) of Rta in Dox-treated TW01TetER
The expression of p53, p21, SFN and p27 were increased in Rta induction. Increasing phosphorylation of H2AX was observed on day5 and day6 after Rta induction.
2. Compare the expression level of p21, SFN, c-Myc, p53 and p63 in Dox-treated TW01TetER and TW05TetER
The expression of p53, p21, and SFN were increased in Rta induction while the levels of c-Myc and p63 were decreased.
3. The cytotoxicity of U0126, Rapamycin and LY294002 on TW01TetER
IC50分別為：U0126 40uM, Rapamycin >10uM, LY294002 60uM
4. Test the effect of serum starvation and no glucose on the cell morphology in TW01TetER and TW05TetER
A. 在50000顆細胞的seeding下，serum starvation 48小時可以觀察到細胞生長停滯(也許是緩慢)。
B. 19號長得比16號快 (48小時內活細胞數會較16號多)，但19號也較16號快死(16號活細胞數較19號多)，我覺得19號代謝較快。
C. 不管是TW01或是TW05，細胞缺少葡萄糖最後會死亡(在第五天觀察)。而在缺乏葡萄糖48小時的情況下再添加葡萄糖回去，細胞可以回復正常生長。
D. Serum與Glucose對細胞的生長都很重要。

RNA-Seq Data USB 1 and 2

2011/07/06_
由 sufang 在 三, 07/06/2011 - 00:00 發表 Older Posts BrCa Dan Robinson RNA-seq USB

2011-07-06

2012-04-09

06/29 Lab Meeting-小嬿

1. 修正sonication條件為30" ON, 30" OFF, 8 cycles, 利用此條件在TW01TetER_16細胞做ChIP. 初步測試myc primer, PCR條件未達到最好: 1) 跑膠結果有很多用剩的primer或dimer; 2) input量很多, 而IP的量很少, 沒有調整好比例; 3) myc-2和myc-3的product跑2% agarose不漂亮
-->end-point PCR 固定template量, 調整input的跑膠量; myc-2 myc-3跑3% agarose gel
-->試試Q-PCR條件

2. 利用Thermo fractionation kit分出TW01TetER_16細胞的nuclear extract做IP, WB結果看到CTCF有抓到疑似Rta的band, 但用Flag抗體IP則看不到CTCF.
-->將Flag-Luc, ER, KR, gZ送入HEK293細胞中表現24 hrs後收lysate做IP, WB結果看到CTCF可抓到明顯的ER(by 467)和微弱的gZ(by 4F10), 抓不到Luc(by Flag)和KR(by Yoshi's Ab); 但用Flag IP, 則在四組細胞中都沒有看到CTCF.

To ship RNA sample without dry ice

2011/06/28 To ship RNA sample without dry ice
由 sufang 在 二, 06/28/2011 - 10:19 發表 Older Posts RNA-seq shipping RNA samples

This is an old post from sophia at 仲彥老師's lab (sophia).  2011-06-28
There is a new way to preserve samples. In room temperature, not deep freeze in -80C.

http://www.biomatrica.com/rnastable_products.php (not working)
try this one : tinyurl.com/yasox9n2

The tech sounds interesting and is a perfect case for utilizing the wisdom of nature. 

Anhydrobiosis

"Life Without Water", a natural mechanism that prevents degradation.

Some eukaryotic extremophiles, such as tardigrades and brine shrimp, developed a natural biological mechanism called "anhydrobiosis" that enables their survival while dry for up to 120 years. During these extended time periods, DNA, RNA, proteins, membranes and cellular systems are protected and can be revived by simple rehydration.

Biomatrica's core technology transfers the molecular principles of anhydrobiosis to a synthetic, chemistry-based stabilization technology that works by forming a thermo-stable barrier securely around a sample to provide protection against degradation and loss of biological activity, effectively "shrink-wrapping" the sample until ready to use. Aqueous samples are applied directly to the matrix and air dried at room temperature prior to storage. The air-drying process activates the formation of the protective SampleMatrix^® barrier. Samples are recovered by simple rehydration and are ready for immediate use; sample loss and further purification are thus eliminated.

SampleMatrix^®

SampleMatrix^® — Biomatrica's proprietary core technology platform.

Biomatrica^® has developed a proprietary core technology, SampleMatrix, to stabilize and protect biological materials at room temperature without degradation. The SampleMatrix technology was designed by combining extremophile biology that enables long-term survival of organisms in extremely dry environments with synthetic chemistry. By exploiting these unique characteristics, Biomatrica products enable researchers to stabilize biological samples for long periods of time.

Samples are preserved through formation of a protective thermo-stable barrier during a simple air-drying process. Samples are protected from degradation from heat and UV light. Since SampleMatrix is designed to completely dissolve within minutes during sample rehydration, there is complete and rapid recovery of samples without degradation or loss of biological activity. The recovered samples can be used directly for downstream applications without further purification.

• Stabilize and Store Samples
  Biomatrica's products protect sample integrity, thus increasing reproducibility.
• Just add Water for Rapid and Complete Sample Recovery
  SampleMatrix dissolves completely within minutes and samples can be used directly in downstream applications.
• Use Samples Directly in Downstream Applications
  No purification is required since SampleMatrix does not interfere with downstream assays in rehydrated form.

Start: 06/28/2011

生技醫藥國家型計畫NRPB誠徵生力軍

研究主題: Identification of gene fusions in cancer cells by RNA-seq

1. 碩士級研究助理
2. 博士後研究人員

學歷及專長:
生物資訊、資訊工程或相關研究系所畢業
熟悉perl、python、linux、unix，有分析基因體序列經驗者
具備分子生物學、細胞生物學之專業知識者尤佳

工作內容：本計畫含wet-bench及dry-lab兩種性質的工作。合作團隊包括台大癌症研究中心盧彥伸；國衛院癌研所林素芳；中研院資訊科學研究所林仲彥 (工作地點主要在台北；工作待遇依國科會支薪標準，享勞健保)。

應徵碩士級研究助理請下載並填妥此頁問卷;
應徵博士後研究人員請下載並填妥此頁問卷。

應徵方式：請將 (1) 履歷與填妥之問卷 (2)二位以上推薦人之連絡資料 (3)最高學歷論文摘要，逕寄蘇玉芬小姐(suyufen7011@gmail.com) 或是郵寄至『100台北市中山南路七號台大醫院腫瘤醫學部 』。

資料審查通過者則安排進行面談，資料恕不退件。面談時間及地點將以e-mail或電話通知。

6/22 Lab Meeting-ingrid

(1) 以shLuc、shOGT、shMGEA5的lentivirus (稀釋倍數1/8) 送入ERKV (6x10^5/2ml/well/6well*6) 中，48小時後加Dox，再48小時收細胞計數螢光比例(第4次實驗)。發現綠螢光的細胞佔全部細胞約80-85%；紅螢光的細胞佔綠螢光細胞約30%(Dox)、5-10%(NC)、30%(shLuc)、40-50%(shOGT-A1)及65-70%(shMGEA5-B2)。收lysates進行WB確認。且用IP將KR抓下，用WB(RL2)來確認O-GlcNAc變化程度。
(2) 進行Q-PCR檢查送入shOGT的293FT細胞樣本中，MGEA5的mRNA是否有減少，反之亦然。由於結果無法排除off-target作用，進行或重複實驗以確認。
(3) TW01TetLuc有2株clones及TW05TetLuc有8株clones進行IFA (Flag Ab) 確認。由於背景值太高，無法確定是否有Luc蛋白質表現，故進行WB以確認。

ChIP-Seq皇后網站

Dan介紹的ChIP-Seq皇后網站。 參考一下她的Protocol專區。

0615 Lab meeting-蔡小丸

1. 再次整理Pin1對於K-RTA的影響, 結果顯示Pin1會結合至K-RTA 並且影響K-RTA之穩定性, 而Pin1結合至K-RTA是經由其WW domain, 且isomerase activity也貢獻於pin1與K-RTA之交互作用
2. 此外, S634A/S636A被anti-MPM2結合的能力較差, 表示S634/S636可能是Pin1所結合的位點
3. Pin1對於K-RTA活性的影響 則尚未釐清
4. 完成oral cancer tissue array (OR2081)的DDR1染色

06/08 Lab Meeting- 小夏

1. The long-term kinetics (0-6 days) of Rta in Dox-treated TW01TetER
2. The cytotoxicity of U0126, Rapamycin and LY294002 on TW01TetER
3. Effect of serum starvation on the cell morphology, cell number and cellular hypertrophy

TW01TetER相關

(AC503, Ingrid, 2010-05-18) 6-well plate format (200K/well = 100K/ml)(1) No Dox control of both cells grew similarly before Day4, doubling time約為24h
(2) #7 continued to grow between Day 4 and Day 7
(3) #19 stopped to increase after Day 4 (猜測是第三天換 medium時同時移掉 死細胞)
(4) YLC 將Day 8細胞拿來做SA-b-Gal staining,是positive, 但比例不詳

(AC 263 小夏) 6-well plate format (200K/well = 100K/ml) DNA content analysis @ 48 h
Results: 71% vs. 78% (#7); 72% vs. 84% (#16); 72% vs. 85% (#19)

(AC 278 小夏) 6-well plate format (100K/well = 50K/ml) DNA content analysis @48 h
Results: 49% vs. 57% (#7); 56% vs. 80% (#19)
(AC401, 小嬿) 24-well plate format (10K/well = 25k/ml); 7.5% FBS, 三天補一次Dox
(1) No Dox control of both cells grew fine until Day 6 (i.e. in 24-well plate, 從10K 長到 200/300K都還OK換算為concn就是500K-750K/ml都還可以)
(2) On day 8, both #16 and #19 have dead cells

所以綜合大家的條件若細胞養在 50K/ml, 在前五天估計細胞都不會因太滿而死掉。上圖附上小嬿細心的記數圖表，是八天的結果。

考慮用Bioruptor sonicate你的protein extract

上週末Bioruptor業務幫我們把儀器fix好的同時，也提及許多Lab在置備protein extract的同時，現在都會加一步sonication的步驟。

set at "Low", 5 cycles.

據說這樣才可以充分地把胞膜上面的蛋白質都萃取出來。有做膜蛋白的人要注意一下! 非膜蛋白的人也可以試試看多加這一步驟，protein是不是萃取得更完全。

06/01 Lab Meeting-小嬿

1. 設計12個target gene的CTCF binding site primers. 初步用ERKV及Raji的genomic DNA測試primer, 九組較佳, 三組較弱, 一組沒有p出來. -->後面這四組primer會再做進一步確認condition
2. 利用IP觀察CTCF是否跟Rta有interaction. IP CTCF後有抓到Rta, 但其抓到的量與Dox induction與否沒有關係(偽Rta?). (使用ICON的blocking reagent後, IgG的band有比較乾淨.)
3. 利用Thermo的fraction kit區分TW01TetER-19細胞的cytosol及nuclear protein. PARP1及CTCF乾淨地位於核內; Rta, p53, H3在cytosol有殘留; GAPDH及a-tubulin主要在質; 怪的是PCNA大部分在細胞質.

-->1. 這篇paper指出位於質內的PCNA具有anti-apoptosis的效用(in neutrophil), 或許這是TW01細胞可以serum starvation還如此快樂的原因之一. (而一點點的PCNA在核內就夠DNA replication 用?或大部分細胞在G1 phase, PCNA不用進核工作)
-->2. 試試沛文的fractionation方法
-->3. 試試IP Flag, 看CTCF是否會被抓下來