OSCC Cell Line STR information 20150312 更新版
由 sufang 在 一, 03/30/2015 - 08:45 發表 Oral Cancer OSCC Cell Line STR analysis
Dear all,
更新oral cancer相關細胞株之STR資訊
共有16個細胞株, 其中DOK, FaDu, HOK, HSC-3, KB, OEC-M1, SCC-15, SCC-9, Tu-183(共9株)有送件STR的資訊(詳如附件)
須請大家注意的資訊如下:
1. HSC-3, 於Dr.郭靜娟's lab的新HSC-3為真, 請非此來源者把舊的細胞扔了吧! (p.s Dr. 李岳倫於2014年7月及9月分別定的STR結果同新HSC-3, 推測亦為可用細胞株)
2. KB, 於Dr.郭靜娟's lab定STR結果為HeLa細胞, ATCC/JCRB資料庫也證明KB為HeLa. 請勿使用此細胞株!
3. SCC-15, Dr.李岳倫's lab 於2014年9月及10月分別定的STR結果皆與SAS細胞株相符(90.9% match), 使用此細胞株請小心!
4. Tu-183, 於Dr.江士昇's lab於2014年12月所定STR結果未比對到現今資料庫中任何細胞株, 推測可能為可用細胞株. 但Dr. 江提供資訊:expression pattern與預期不同, 大家使用此細胞株請小心!
以上,
如有問題或錯誤的地方, 敬請不吝指正! 謝謝!
敬祝 研究順利
嬿如 敬上
原檔按我下載 (2015_0320_cell_lines_str_information20150312ding_gao.xlsx)
Mar 29, 2015
2015-03-10 Journal Club Paper
各位老師、學長學姊大家好:
附檔是我這周要報告的paper
請查收 謝謝大家
NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival
附檔是我這周要報告的paper
請查收 謝謝大家
祝 研安
韻涵 敬上
韻涵 敬上
Mar 13, 2015
2015/01/30 國長會議
由 sufang 在 五, 03/13/2015 - 08:29 發表 Pre-published 國長會議 長庚
國衛院長庚雙邊會議 2015/01/30 (五) at 林口長庚
王陸海: miR491-3p is responsible for oral cancer metastasis
葉大森: bile duct cancer (miRNA 200下降), liver cancer (miRNA 200 上升，which is correlated with EMT)
陳鈴津教授介紹 閻紫宸教授另外一個專長是 radio pharmatheutical research (新藥開發 GMP等等)
-----------------------
趙盈凱 副教授【胸腔外科】可吸收性生物醫材在食道癌之應用
東方人食道癌 是由ABC引起的、西方的食道癌則多由胃食道逆流
證實CCRT+Surgert is better therapy than just surgery
-Big Database (2400 病人 由該醫師和助理慢慢build up)
-健保加值資料庫
-cisplatin membrane
biodegradable polymers in thoracic disease
-----------------
陳立宗 特聘研究員兼所長 【國衛院癌症研究所】 Pancreatobiliary Cancer Clinical Trial in Taiwan
–TCOG很多東西需要長庚醫院幫忙 e.g. 陳敏夫教授 adjuvant interferon alpha 2b TCPG T1297 (this is a negative study, indicating that Taiwanese surgery was very successful)
–原來文獻建議 肝癌患者 開完刀後若能 加上抗病毒藥物治療 可有效降低recurrence rate.
但是由TCOG的結果看來, not necessary.
-----------------
葉大森: bile duct cancer (miRNA 200下降), liver cancer (miRNA 200 上升，which is correlated with EMT)
陳鈴津教授介紹 閻紫宸教授另外一個專長是 radio pharmatheutical research (新藥開發 GMP等等)
-----------------------
趙盈凱 副教授【胸腔外科】可吸收性生物醫材在食道癌之應用
東方人食道癌 是由ABC引起的、西方的食道癌則多由胃食道逆流
證實CCRT+Surgert is better therapy than just surgery
-Big Database (2400 病人 由該醫師和助理慢慢build up)
-健保加值資料庫
-cisplatin membrane
biodegradable polymers in thoracic disease
-----------------
陳立宗 特聘研究員兼所長 【國衛院癌症研究所】 Pancreatobiliary Cancer Clinical Trial in Taiwan
–TCOG很多東西需要長庚醫院幫忙 e.g. 陳敏夫教授 adjuvant interferon alpha 2b TCPG T1297 (this is a negative study, indicating that Taiwanese surgery was very successful)
–原來文獻建議 肝癌患者 開完刀後若能 加上抗病毒藥物治療 可有效降低recurrence rate.
但是由TCOG的結果看來, not necessary.
-----------------
Mar 6, 2015
ds-cDNA prep for gene fusion detection
3/6/2015 DR 提醒
1: Daw-Yang seems to use Clontech ds-cDNA prep for single cell Seq + Tagmentation
2: There is no way to add specific primer in this way...
According to Daw-Yang: I will use Nexera XT for DNA prep.
Figure 1 AMP for targeted RNA and DNA sequencing (pdf 4231).
Double-stranded cDNA (dscDNA) synthesis starts with total nucleic acid or RNA from fresh or FFPE material without ribosomal RNA or genomic DNA(gDNA) depletion. Solid-phase reversible immobilization (SPRI)-cleaned double-stranded cDNA or fragmented genomic DNA is processed with end repair and dA tailing, directly followed by ligation with a half-functional adapter. SPRI-cleaned, ligated fragments are amplified with 10–14 cycles of multiplex PCR1 using gene-specific primers (GSP1 pool) containing a PCR multiplexing tag (brown) and a primer complementary to a portion of the universal ligated adapter (short green). An unknown fusion partner or target sequence is indicated by a question mark. SPRI-cleaned PCR1 amplicons are amplified with a second round of 10-cycle multiplex PCR2 using a combination of GSP2 pool nested gene-specific primers (3' downstream of GSP1), which are tagged with the second adapter sequence specific for Ion Torrent (black-blue) or Illumina (black-red, subsequently tagged with an indexing primer (red-orange-purple)), and a second nested primer against the ligated universal adapter (long green). After a final SPRI cleanup, the target amplicon library is ready for quantitation, downstream clonal amplification and sequencing.
Fluidigm 4-Primer Amplicon Tagging
The Access Array 4-primer amplicon tagging scheme is based on a step-out four primer PCR strategy where tagged target-specific (TS) primer pairs are combined with sample-specific primer pairs that contain a barcoding sequence and the adaptor sequences used by Illumina sequencing systems. By incorporating sample-specific barcodes, all 2,304 PCR products generated from a 48.48 Access Array Integrated Fluidic Circuit (IFC) are unique and can be pooled together to run in a single sequencing experiment.
Nextera_xt_sample_prep
Feb 27, 2015
TCGA HNSCC Marker Paper
由 sufang 在 五, 02/27/2015 - 10:08 發表 Oral Cancer HNSCC TCGA
Comprehensive genomic characterization of head and neck squamous cell carcinomas (pdf 4292; PubMed Link)
The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.
Five Figures in the Main Text:
Figure 1 | DNA copy number alterations. a, Copy number alterations by anatomic site and HPV status for squamous cancers. Lung squamous cell carcinoma (LUSC, n = 358) and cervical squamous cell carcinoma (CESC, n = 114). b, Unsupervised analysis of copy number alteration of HNSCC (n = 279) with associated characteristics. The rectangle indicates chromosome 7 amplifications in the purple cluster. NA, not available.
Figure 2 | Significantly mutated genes in HNSCC. Genes (rows) with significantly mutated genes (identified using the MutSigCV algorithm; q , 0.1) ordered by q value; additional genes with trends towards significance are also shown. Samples (columns, n = 279) are arranged to emphasize mutual exclusivity among mutations. Left, mutation percentage in TCGA. Right, mutation percentage in COSMIC (‘upper aerodigestive tract’ tissue). Top, overall number of mutations per megabase. Colour coding indicates mutation type.
Figure 3 | Candidate therapeutic targets and driver oncogenic events. Alteration events for key genes are displayed by sample (n = 279). TSG, tumour suppressor gene.
Figure 4 | Integrated analysis of genomic alterations. a,b,Samples (n=279) ared isplayed in columns and grouped by gene expression (a) or methylation (b) subtype (sub.). Unadjusted two-sided Fisher’s exact test P values assess the association of each genomic alteration. Methylation probe location of CpG islands, shores and shelves are shown on the left of b. Annotation shows HPV status and subtype (16, 33 and 35). CN, copy number.
Figure 5 | Deregulation of signalling pathways and transcription factors. Key affected pathways, components and inferred functions, are summarized in the main text and Supplementary Information section 7 for n = 279 samples. The frequency (%) of genetic alterations for HPV(–) and HPV(+) tumours are shown separately within sub-panels and highlighted. Also see Supplementary Fig. 7.15. Pathway alterations include homozygous deletions, focal amplifications and somatic mutations. Activated and inactivated pathways/genes, and activating or inhibitory symbols are based on predicted effects of genome alterations and/or pathway functions.
Ten Parts of Supplementary Information:
S1: Biospecimen collection and clinical data
S1.1. Biospecimen collection and clinical data
S1.2. HPV detection methods
S1.3. Survival analysis **
Figure S1.1. HPV status as a function of clinical and molecular characteristics
Figure S1.2. Receiver operating characteristic (ROC) curves in HPV-associated miRNAs in oropharyngeal HNSCC
Figure S1.3. DNA methylation signatures of HPV
Figure S1.4. Survival analysis for select clinical and genomic variables
Figure S1.5. Survival analysis for platform-specific subtypes
Table S1.1. Summary of clinical data
Data file S1.1. Data freeze clinical dataset (This file contains clinical and demographic information for all patients)
Data file S1.2. Summary of HPV detection results (This file contains the results of molecular analyses used to determine the HPV status of all patients. These include data from in situ hybridization, p16 staining, RNA and DNA sequencing, and the MassArray assay.)
Data file S1.3 Mutation signatures by HPV status (This file contains counts of base changes seen in APOBEC and smoking mutation signatures for HPV(+) and HPV(-) subjects. Fisher’s exact test p-values are also shown.)
S2: Copy number analysis
S2.1. SSNP array-based copy number analysis
S2.2. Structural alterations
Figure S2.1. GISTIC 2.0 analysis of significantly reoccurring focal alteration in 279 HNSCC tumors
Figure S2.2. GISTIC amplification and deletion peaks in lung squamous cell and cervical squamous cell carcinoma
Figure S2.3. Comparison of GISTIC 2.0 analyses of 243 HPV(–) and 36 HPV(+) head and neck tumors
Figure S2.4. Number of copy number segments in HPV(+) and HPV(–) samples
Data file S2.1. GISTIC amplification and deletion peak annotation in head and neck squamous cell (all samples, by HPV
status, and by site), lung squamous cell carcinoma, cervical squamous cell carcinoma (results from GISTIC analyses for head and neck (HNSC), lung, and cervical squamous cell carcinoma. Additional analyses for HNSC were conducted by HPV status and tumor site.)
Data file S2.2. Fisher’s exact test p-values for frequency comparisons of significantly reoccurring alterations by HPV
status and site (results of analyses of frequency comparisons of significantly reoccurring DNA copy number alterations by HPV status and tumor site. Fisher’s exact test p-values are also shown.)
S3: RNA sequencing
S3.1. RNA sequencing and expression quantification
S3.2. RNA-Seq for confirmation of somatic alterations reported in whole exome sequencing
S3.3. Gene fusion detection
S3.4. RNA-Seq for gene splicing and viral integration
Figure S3.1. RNA-Seq for confirmation of somatic alterations reported in whole exome sequencing
Figure S3.2. FGFR3-TACC3 fusion event
Figure S3.3. EGFR vIII mutant sample
Figure S3.4. Exon 14 skipping in MET+
Figure S3.5. Alterations of CDKN2A gene structure, copy number, and expression of its protein coding transcripts p16INK4A and p14ARF
Figure S3.6. Integration of DNA mutation type, copy number, and gene expression for CDKN2A+
Figure S3.7. Alterations of FAT1 gene structure, copy number, and expression
Figure S3.8. Integration of DNA mutation type, copy number, and gene expression for FAT1+
Figure S3.9. Integration of DNA mutation type, copy number, and gene expression for predicted driver genes relevant to HNSCC
Figure S3.10. Distribution of HPV integration breakpoints across the host genome
Figure S3.11. The KLK12 gene documents recurrent alternate transcription in HNSCC Figure S3.12. Heterogeneous TP63 isoform usage in HNSCC+
Data file S3.1. RNA-Seq predicted fusions ( contains the results of fusion detection analyses performed with MapSplice. )
Data file S3.2. Viral integration sites (information about HPV viral integration sites based on the analysis of RNA and DNA sequencing
data.)
Data file S3.3. SigFuge clustering results for alternatively spliced genes (the results of SigFuge analyses to detect differential expression of multiple gene isoforms. Uncorrected p-values are shown.)
Feb 17, 2015
Molas Analysis on Arecoline-Treated HOK Cells
02/16/2015
MTHFR, SHMT1, SHMT2
02/17/2015
1. Library comparison
2. OKB2/AC (>=0.1) combined with OKB2/untreated (>=1), >= 2-fold (2886 transcriptid), sent to GO
Biological Process (BP): GOBP
Molecular Function (MF): GOMF
Cellular Compartment (CC): GOCC
03/28/2015
Excel 製圖時
1. On "OKB2" Filter > 3, shade them
2. On "Fold OKB2" Filter >=1.5 shade with red; Filter <=0.67 shade with blue
3. On "S1F" Filter > 3, shade them
4. On "Fold S1F" Filter >=1.5 shade with red; Filter <=0.67 shade with blue
5. On "THP1" Filter > 3, shade them
6. On "Fold THP1" Filter >=1.5 shade with red; Filter <=0.67 shade with blue
04/02/2015
感謝大家的製圖與整理! 再來要做啥? 我有幾個想法
1. Nucleosome assembly的原料, i.e. histones, 全面下降. 表示細胞若正在S phase, 再來的nucleosome assembly將有巨大變化. 是符合hypomethylation嗎?
2. 我們map出來的histone gene多在ch6, 少數幾個在ch1. 這些cluster在一起的gene family, 使要被調降的?
3. 還有沒有其它類似例子?
4. 如果ROS是元凶 (許多AC造成的上升或下降, 可被GSH reverse 回來), 機制是什麼?
5. To confirm or extend more experiments, let's try on OKF4-hTert, NP460hTert and CGHNK2.
04/14/2015
1. Focus on nucleosome downregulation: histone cluster 1 on ch6 and histone cluster 2 on ch1.
2. Large hypo-methylation blocks: 在正常細胞時多為LAD/LOCK domain, 經過"transformation stimuli"後, 成為hypo-methyl region.
LAD: lamina-associated domain
LOCK: laroge organized chromatin lysine-modification
3. 40T/N methylation array中, delta beta >= 0.35 定義為 hypermethylated genes, delta beta <= -0.3 定義為 hypomethylated genes.
4. 40T/N methylation array中:
N_MAX-N_MIN delta beta >= 0.3 計有6830 (表示 normal tissue中, methylation差異性的存在)
T_MAX-T_MIN delta beta >= 0.3 計有10808 (表示 tumor tissue中, methylation差異性的存在)
T AVB-N AVB delta beta >= 0.3 計有702 (Illumina廠商根據其它條件 例如是否有CpG island, 再縮減成37個up, 37個down.
T AVB-N AVB delta beta >= 0.3 計有702 (Illumina廠商根據其它條件 例如是否有CpG island, 再縮減成37個up, 37個down.
5/26/2015 New
On Molas portal:
step 1: OKB2 > 1, OKB2_AC > 0, Fold >= 2, 得 3220 transcripts.
step 2: OKB2 > 0, OKB2_AC >= 2, Fold >= 2 , 得1460 transcripts.
step 3: combine step 1 and step 2, 用 "IF" 公式 , 得 unique transcrpt n = 3563.
step 4: Sort for value, < 0.1者全部replace為0.1, 計算Fold.
step 5: Sort for GeneName / OKB2值大到小 / OKB2_AC 值大到小, 用"IF" 公式過濾unique gene name.
小計: unique gene name n= 2547 (down 1873, up 674).
unique transcript / no gene name n=155.
2547 + 155 = 2702 = 1987 down, 715 up.
Among the 2547 unique genes, we will focus on
(1) chromatin organization genes
(2) cell cycle related genes
(3) TP53 related genes (TP53值較高的transcript 因未達2倍差異, 反未被挑出 )
(2) anti-oxidant genes
(3) secreted proteins
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
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
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
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
Cc: 蔡佳龍
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
Cc: SuFang Lin
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
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
Cc: Angel Chao
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
-------------------------
SuFang
-------------------------
蔡佳龍
To: SuFang Lin
Cc: Angel Chao, 王子豪醫師
To: SuFang Lin
Cc: Angel Chao
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
Cc: 英潔YCK
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
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
Cc: 王子豪醫師
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
To: SuFang Lin
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
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
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
Cc: 蔡佳龍
Bcc: 英潔YCK
Dear Dr. Wang,
Are you talking about MTA thing? 據我所知, SW780的 gene fusion至少有兩篇
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
Cc: 林素芳
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
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
4. 14500rpm, 15min
5. 70% EtOH(cold) 100ul wash 6. 14500rpm, 6min
7. Dry pellet
8. Resuspend in RNasefree H2O 9. Nanodrop
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)"
To: 柯英潔
Cc: 林素芳
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
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)"
Cc: 林素芳
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
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
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)"
To: 柯英潔
Cc: 林素芳
Dear 英潔
寄來的 RNA sample 已收到了 謝謝!
寄來的 RNA sample 已收到了 謝謝!
陳華鍵
------------------
Jan 27, 2015
Oligo檔案區
目前想到的方法如下，請大家集思廣益讓「編號引子」成為正確有效又快樂之工作! 謝謝大家~
Step 1: 拉到本文下方第一則回應、看看最新一個人引子的LAB ID是用到第幾號了, 記住你要開始的號碼.
Step 2: 利用本文右下方「增加新的回應」
Step 3: 主題部分標入你login時間及大名
Step 4: 回去你的Excel檔做編輯
**Excel檔來自生工 : (1) 在最前面加一欄 LAB ID (2) 輸入剛剛記住的號碼並完成編號 (3) copy A to G的資料 paste在ELN回應內文. (4) 記住設定table大小為700 (px) (範例見下面2015/01/01小嬿)
**Excel檔來自每得IDT : (1) 在最前面加一欄 LAB ID (2) 輸入剛剛記住的號碼並完成編號 (3) Move "Tm"、"Final nmoles"、"H2O to 100 uM"、"product bp" up to "Sequence". copy A to H的資料 paste在ELN回應內文. (4) 記住設定table大小為700 (px) (範例見下面2015/01/23 Ingrid)
建議大家自己有一個Excel檔，專門存放oligo sequences..
ps. 範例2015/01/01 小嬿那一筆的LAB ID是瞎編的，最後會刪掉; 2015/01/23 Ingrid這一筆是真的，請下一個幸運兒從978開始使用! Thanks.
2015-02-10 Journal Club Paper
Dear all:
這是我下週要報告的paper
題目為:Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration
敬請多多指教
謝謝
思涵 敬上
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
Sat, Dec 6, 2014 at 9:10 PM
To: Su-Fang Lin Sun, Dec 7, 2014 at 8:17 AM
#1由 sufang 在 三, 01/21/2015 - 15:23 發表。
Regarding sample request at TLCN
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
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.
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
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
Regarding sample request at TLCN
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