Our Thoughts (桀靜精微雷風相薄): Gene Fusion

Showing posts with label Gene Fusion. Show all posts

Feb 13, 2015

寄送SW780 RNA給長庚及行動基因陳華鍵

寄送SW780 RNA給長庚及行動基因陳華鍵
由 sufang 在五, 02/13/2015 - 15:20 發表 Cholangiocarcinoma GeneFusion

From: SuFang Lin [mailto:sflin1@gmail.com] Sent: Friday, January 30, 2015 2:06 PM
To: angel945@cgmh.org.tw
Subject: this is SuFang's email

Dear Dr. 趙:
This is my email. I will prepare ~510 micrograms RNAs from SW780 cells for

your control. Please let me know to whom and to where should I sent. Thanks!

Best, SuFang

--------------------------------------

Angel Chao Fri, Jan 30, 2015 at 2:49 PM To: SuFang Lin
Cc: 蔡佳龍 , knoxtn@cgmh.org.tw

Dear Prof. Su‐Fang,

Thank you very much indeed for your prompt e‐mail. Dr. Tsai (蔡佳龍博士)will give you the details for RNA transfer to our lab. Thanks again and nice meeting you at this fruitful CGMH‐NHRI meeting.

Regards, Angel

-----------------------------------------

蔡佳龍 Fri, Jan 30, 2015 at 4:16 PM To: Angel Chao
Cc: SuFang Lin , 王子豪醫師

林老師您好: 非常感謝您願意提供給我們SW780 cell RNA

由於RNA運送須以乾冰低溫運送比較麻煩不知能否冒昧的跟老師要SW780 cell, 以常溫運送我們培養後再自行抽取RNA 比較不佔用您的助理太多時間

再次感謝老師的幫忙敬祝

研安

蔡佳龍敬上桃園市龜山區復興街五號

林口長庚醫院兒童醫療大樓J棟3樓婦產部實驗室蔡佳龍
電話:0332812008738或8784

[Quoted text hidden]

SuFang Lin Fri, Jan 30, 2015 at 5:10 PM To: 蔡佳龍
Cc: Angel Chao , 王子豪醫師

Dear 佳龍:

因為我們實驗室主要是做頭頸癌, SW780膀胱癌細胞當初是養幾代抽完RNA後就沒有再maintain, 所以送細胞和送RNA是一樣得用乾冰的. 另外是 SW780我是和人要來的, 得要再寫信去國外友人處問可不可以分讓,有MTA問題, 反而比較麻煩。

乾冰運送RNA我有經驗、事實上半年前高醫有人跟我要SW780 RNA,目的與你們一樣,也是當positive control, in which the results looked good. 所以我才有信心跟趙醫師說沒問題.倒是FGFR3的fusion partner很多、即便同一個partner 斷點也不同, 因此若欲以RTPCR 方式篩選的話, 這點要小心.

Best,
SuFang
-------------------------

蔡佳龍
To: SuFang Lin
Cc: Angel Chao , 王子豪醫師

林老師您好: 謝謝的您幫忙與提醒

再麻煩老師了謝謝您

佳龍敬上

---------

SuFang Lin Tue, Feb 3, 2015 at 2:48 PM To: 蔡佳龍
Cc: 英潔YCK , 玉蓮YLC

Dear 佳龍,
一切順利的話我們應該明天星期三就可寄出冷凍包裹, 我們會請黑貓宅急便送, 標

示貨到付款, 寄到桃園市龜山區復興街五號

林口長庚醫院兒童醫療大樓J棟3樓婦產部實驗室這樣子OK嗎? 對了,可能還需要填你們實驗室電話. 謝謝!

Best, SuFang

---------------------

蔡佳龍 Tue, Feb 3, 2015 at 2:55 PM To: SuFang Lin
Cc: 王子豪醫師 , 趙安琪醫師

林老師您好: 我們實驗室的電話

0332812008784或8738 我的手機0975510816 再次感謝您的幫忙

佳龍敬上

---------------

蔡佳龍
To: SuFang Lin Cc: 王子豪醫師

Wed, Feb 4, 2015 at 7:27 AM

林老師您好: 我本身是在長庚醫院基因體核心實驗室工作,主要以affymetrix晶片研究基因體

的變化。
目前affymetrix新晶片human transcriptome array (HTA)號稱可以偵測alternative splicing與fusion gene。不知能否跟老師多要些sw780 RNA(10ug)讓我們可以測試晶片能力,我們也會將晶片資料與老師分享。
再次麻煩老師

謝謝您的幫忙佳龍敬上

----------

SuFang Lin Wed, Feb 4, 2015 at 8:45 AM To: 蔡佳龍 , 王子豪醫師
Bcc: 玉蓮YLC

Dear 佳龍,

其它cell line的RNA可以嗎? 昨天我們有多分出一管OSCC cell line TW2.6的RNA (10 ug 多一點點). TW2.6也有fusion gene, 但不是FGFR3, 而是一個receptor protein tyrosine phosphatase.

因為我們習慣讓分裝好的RNA過夜冷凍, 隔天再寄. 所以要給你們的SW780 RNA昨天已分裝好冰回. Tenug 分一點過去做FGFR3的RTPCR, 剩下的做HTA應該很足夠喔! How do you think?

SuFang

-----------------------

王子豪 ReplyTo: 王子豪

Dear Dr. Lin:

Wed, Feb 4, 2015 at 9:20 AM

Thanks for the reply. If you permit, we will analyze the SW780 RNA with HTA microarrays too and report the results to you as soon as we get them.

Best regards, TzuHao

---------------------------------

SuFang Lin Wed, Feb 4, 2015 at 9:36 AM To: 王子豪
Cc: 蔡佳龍
Bcc: 英潔YCK , 玉蓮YLC

Dear Dr. Wang,
Are you talking about MTA thing? 據我所知, SW780的 gene fusion至少有兩篇

paper都有發表了, 所以我覺得OK.

上週佳龍問我要cells時,那就會有concern,因為我也是跟友人要來的,要分讓給你們, 我的習慣是還會請你們跟他簽一次MTA. 想說如果只是做control, 給RNA就可避掉這些麻煩啊.

Thus, go ahead and use both TW2.6 and SW780 RNA for your HTA analysis. 各 10ug 應該夠吧, 還是你們需要更多, 那我們就要再回溶一次SW780, 延遲到明天寄 > Which is also fine with us.

Best, SuFang

------------------

柯英潔 Wed, Feb 4, 2015 at 4:35 PM To: "hcchen@actgenomics.com"
Cc: 林素芳 , 陳玉蓮

陳博士您好

今天下午已經將RNA樣本用黑貓冷凍寄送給您預計明天中午前會收到請依照以下步驟沉澱RNA使用

每管約為12ug RNA溶於formamide,體積20ul,根據以往經驗沉澱後回收約80% 以上,因此最後會得到約10ug的RNA。
沉澱步驟為
1. add 1/10V 3M sodium acetate

2. add 3V EtOH(Merck), mix well 3. 20°C O/N
4. 14500rpm, 15min
5. 70% EtOH(cold) 100ul wash 6. 14500rpm, 6min

7. Dry pellet
8. Resuspend in RNasefree H2O 9. Nanodrop

 如有任何問題再跟我聯繫
 謝謝

柯英潔分機35122 林素芳老師實驗室癌症研究所國家衛生研究院

----------------

Hua Chien Chen (陳華鍵)

To: 柯英潔
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

Dear 英潔
謝謝你寄來的 RNA 以及後續處理相關資訊,我會請公司同仁依此實驗流程操

Wed, Feb 4, 2015 at 4:45 PM

作。謝謝!

陳華鍵

[Quoted text hidden]

This email may contain confidential information of ACT Genomics Co., Ltd. If you are not the addressee, please notify the sender and delete this email immediately. Any unauthorized review, use or distribution by anyone other than the intended recipient is strictly prohibited.

柯英潔 Wed, Feb 4, 2015 at 4:48 PM To: "Hua Chien Chen (陳華鍵)"
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

陳老師您好

我想再補充二點
1. RNA在formamide中很穩定,如果還沒要往下做,直接丟 80C冰箱冷凍起來。 2. 步驟中,20度沉澱只需要1小時以上即可

柯英潔分機35122
林素芳老師實驗室
癌症研究所
國家衛生研究院

----------------------

Hua Chien Chen (陳華鍵)

To: 柯英潔
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

Dear 英潔
寄來的 RNA sample 已收到了謝謝!

陳華鍵

------------------

Jan 21, 2015

Fusion gene detection 2014-Dec 及 TLCN檢體申請

Fusion gene detection 2014-Dec 及 TLCN檢體申請
由 sufang 在三, 01/21/2015 - 11:29 發表 Cholangiocarcinoma cholangiocarcinoma FGFR2 gene fusion

Nov 27, 2014 at 2:57 PM

<910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Dear SF,

Only minor suggestions (in red color). Are you going to list the oncogenes screening in your proposal or we can just perform the exam?

I am current reading most recent driver mutations in the cholabgiocarcinoma, but I will be great appreciated if you can also give me the most updated/recurrent mutated genes in cholangiocarcinoma. Than I will design primers for those candidate genes.

Furthermore, I do not have a MiSeq in my lab yet, so I outsourcing now. I wish

I can get my own MiSeq machine in one year.

Kindly regards, Daw-yang

Thu, Nov 27, 2014 at 4:34 PM

Su-Fang Lin To: Daw-yang Hwang

Three references for you! Best, Su-Fang

4001_2014_03_New Routes to Targeted Therapy of Intrahepatic Cholangiocarcinomas Revealed by Next-Generatiion Sequencing.pdf

4239_2013_12_Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection- related bile duct cancers.pdf

3970_2013_12_Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas.pdf

Daw-yang Hwang <910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Please see the attached file for the updated TLCN form. Daw-yang

about your application

6 messages

To: "sflin1@gmail.com"

Dear Dr.Lin,

Mon, Dec 1, 2014 at 7:22 PM

Welcome to apply for TLCN samples. However, I need to tell you that many of the cholangiocarcinoma samples are not so good in the RNA quality (due to tumor necrosis), and many have low tumor %. Thus, though we have more than 150 cases, but it is impossible to get 96 cholangiocarcinoma with high tumor % and good RNA quality for your application. we probalbly can give you around 40-50 cases. If you just need DNA and tissue section, it may be possible.

shiufeng

Su-Fang Lin

To: <910208 mail.kmuh.org.tw="">

Dear Daw-Yang,

Mon, Dec 1, 2014 at 9:05 PM

What do you think? Can I tell her that for our purpose not so ok RNA are still worth a try? Thank you very much! SF

Sent from Gmail Mobile

Daw-yang Hwang <910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Dear SF,

Tue, Dec 2, 2014 at 5:06 AM

I can understand the RNA quality issue. We can try 48 RNA samples first to see how it goes. For DNA, it can be as much as possible, so maybe we should apply 144 DNA samples?

Daw-yang

Su-Fang Lin Tue, Dec 2, 2014 at 9:37 AM To: ,

Dear Dr. Huang,

Thank you very much for telling us the tumor necrosis/poor RNA quality issue. With this regard, we would like to apply 48 RNA samples and associated unstained paraffin sections; and 144 DNA samples of cholangiocarcinoma tissues. A new application form with revised case numbers is attached in the mail.

Thank you very much for your great help!!

Best, Su-Fang

TLCN sample_request_NICR_LinSF v2.pdf 282K

To: Su-Fang Lin ,

144 is too many. Please reduce to 80 to 90 cases.

Do you accept cases with a tumor percentage around 50 percent only? iPhone

Su-Fang Lin To:

Tue, Dec 2, 2014 at 12:25 PM

114太多啊! 真對不起，因為機器一次跑48個所以想說取它的倍數來申請...

好，那我們就申請九十例DNA檢體。另外，50% tumor cellularity 應該是OK的。謝謝妳!

素芳

Re: about your application

9 messages

Sat, Dec 6, 2014 at 9:10 PM

To: Su-Fang Lin Cc:

Please remember to submit a revised form of LOI.

Su-Fang Lin Sat, Dec 6, 2014 at 10:19 PM
To: ,
Dear Dr. Huang,
Enclosed please find my revised LOI.

Again, many thanks for all your advice and help!

Best, Su-Fang

Letter of Intent of TLCN_NICR_SuFangLin v3.pdf

 To: Su-Fang Lin Sun, Dec 7, 2014 at 8:17 AM

You did not change the case number for DNA. Many ICC tumors have low DNA tumor %, and need to use laser capture microdissection, which will be quite expenstive (1000/ 1ug). Anyway, the total number still can not reach 144 cases. Could you reduced to 96 cases (48 wells x2) for DNA?

shiufeng

Su-Fang Lin To: Sun, Dec 7, 2014 at 10:00 AM

咦，我在昨天LOI (v3)有改成90例DNA檢體及50% tumor cellularity. 是還有地方漏掉、沒改到的嗎、還是檔案沒有附檔成功? 讓我再送一次! 麻煩您再檢查一下，不好意思。

Best,

Su-Fang

sfhuang@nhri.org.tw To: Su-Fang Lin Sun, Dec 7, 2014 at 8:42 PM

You only made change in the Item V, but you did not change in Item III, No.2, which is the most important part. I can not revised the LOI for you, since it is in pdf file.

shiufeng

Su-Fang Lin Sun, Dec 7, 2014 at 9:03 PM To:

真是糊塗，不好意思. Best, Su-Fang

sfhuang@nhri.org.tw Sun, Dec 7, 2014 at 9:11 PM

To: Su-Fang Lin

OK. It will be sent out for review.

Shiufeng

Su-Fang Lin To:

Great, thank you very much, Dr. Huang!

sfhuang@nhri.org.tw To: Su-Fang Lin Sun, Dec 7, 2014 at 9:19 PM

you are welcome.

Shiufeng

#1由 sufang 在三, 01/21/2015 - 15:23 發表。
Regarding sample request at TLCN

Nov 22, 2014

Fusion gene detection

由 sufang 在六, 11/22/2014 - 09:01 發表 Cholangiocarcinoma cholangiocarcinoma FFPE Gene Fusion

Nov 5 2014

Daw-yang Hwang 黃道揚 to LTCHEN, yngmiin

Dear Prof Lin, Dr. Chiang, and SF:

I designed primers for ALK, ROS-1 FGFR2, FGFR3, and I can add primers for RET.
In my research part (bladder and upper urinary tract cancer), I am collaborating with urologists in KMUH. Hopefully I can get cDNA samples from then late this month. I can run the above panel for cholangiocarcinoma, oral cancer, or any cancer samples you are interested. I am currently testing some FFPE samples.
Brief, the protocol:
1. Samples (fresh or FFPE)
2. Extract RNA
3. RT-PCR for single-starnded cDNA
4. second cDNA systhesis (double-stranded cDNA)
5. Nextera XT kit
6. Specific PCR amplification
7. Barcode PCR (up to 288 different samples in one MiSeq run)
8. Purification
8. MiSeq run

Maybe we can meet sometimes to have a discussion, for:
A) where to run MiSeq
B) discuss whether we want to include potential fusion on both ends or run known fusion-direction.

Kindly regards,
Daw-yang

A company developed similar kits this year:
http://www.enzymatics.com/archer/

Nov 5 2014

Su-Fang Lin to 陳所長

Dear 所長:

那我就向黃秀芬醫師申請TLCN的cholangioncarcinoma sample cohort做:

1) FGFR2 IHC 染色 (我們剛剛買了一些commercial的tissue array, 在測試抗體條件)

2) cDNA for 道揚's RT-PCR test.

相請教您的是，用您的名字申請? 還是用我的?

Best,

Su-Fang

Nov 6 2014

Su-Fang Lin to Daw-yang Hwang., LTCHEN

Dear Daw-Yang:

Congratulations to your 104 NHRI grant!!! 我先押寶FGFR3 gene fusions in your bladder and upper uinary tract unrothelial cancer! (by the way, we have just completed FGFR3 IHC staining for some commercial tissue arrays, in case you need it someday, please let me know :)

Su-Fang 敬賀

Nov 6 2014

Daw-yang Hwang 黃道揚 to me

Dear SF,

Thank you for your great help, I plan to run several tyrosine kinase fusion genes together in the bladder cancer samples. Which organ origins are those commercial samples?
Let me know if you want to test your oral cancer (or other cancers) samples. I will run in-house designed oncogene panel for my bladder cancer, using Fluidigm-MiSeq plateform.

Talk to you soon,
Daw-yang

Nov 6 2014

Su-Fang Lin to Daw-yang Hwang

I remember we did include some bladder cancer tissues , please check this post
http://eln.nhri.org.tw/lims/?q=node/1798 (還記得你的user name/password嗎? DYH/DYH123)

I will call you to discuss more about FGFR3 gene fusions in oral cancer. 你今天何時比較有空? 11點OK嗎? 或是下午、或是明天你可以的時間! 謝謝。 (對了，請再給我一次你office電話)

Nov 6 2014

Daw-yang Hwang 黃道揚 to me

I will check my user name in the NHRI system.

You can call me at 11AM, 07-3121101 x 2266 (this is the lab, I will be there at 11AM)
(Cellular phone is 0932351464 or 0975356113 if case you need them)

Talk to you soon,
Daw-yang

Nov 6 2014

Su-Fang Lin to Daw-yang Hwang

OK, I will call you at 11AM (lab phone 我比較會講話)
剛剛那個post 連結請用 DYH (user name), DYH123 (password) 登入。與NHRI account 無關喔。

Best,

Su-Fang

Nov 6 2014

Daw-yang Hwang 黃道揚 to me

Dear SF,
Below is the estimated cost of my fusion gene analysis:

A. The cost for known FGFR2 fusion gene detection is minimal:
1. primer (some cost, < NTD 5000)
2. RNA extraction (no cost in NHRI)
3. RT-PCR (some cost)
4. Run gel for positive band
5. Sanger sequencing band in 4. (some cost)
Total cost should be less than NTD 10000

For unknwon TK fusion (including FGFR2-fusion, and all other TK candidates) (48 samples)
1. primer (no cost, since I am going to order these primers anyway)
2. RNA extraction (no cost in NHRI)
3. RT-double stranded cDNA (second stranded cDNA kit 10000)
4. Nextera XT library kit (~20000)
5. fusion specific PCR (Fluidigm plate 20 000)
6. Barcode PCR (free from my previous lab)
7. MiSeq (NTD 60 000)
Total cost for run 48 samples in all possible fusion genes is NTD 110,000, 96 samples will be NTD 160,000 (if still use one MiSeq kit)

For your references, the cost for RNA-Seq (rRNA-depletion, minimal 10M read; we get 15M reads) is ~NTD 28000 (威健 OR 基米 has similar prices).

Regards,
Daw-yang

Nov 6 2014

Su-Fang Lin to Daw-yang Hwang.

Thanks a lot for the valuable info! And sorry I had been in a meeting whole afternoon, in case you had called me.

Best,
Su-Fang

Start: 11/05/2014