Fusion gene detection

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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
11/05/2014

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