Mar 20, 2014
Mar 18, 2014
Dan_YiMi
2014/03/18_Dan_YiMi
由 sufang 在 五, 04/25/2014 - 09:39 發表 Pre-published Dan Robinson RNA-seq
由 sufang 在 五, 04/25/2014 - 09:39 發表 Pre-published Dan Robinson RNA-seq
SFL,
I had not heard that news. I am very doubtful about this. We find new fusions i.e. new ETS 5’ partners now and then, but something completely novel detected only by deep sequencing does not smell right.
Functional driving fusions are nearly always expressed at a level that generates dozens to hundreds of fusion reads by our standard sequencing. Read-throughs, tandem duplications and other subclonal events are expressed at much lower levels, the deeper you sequence the more subclonal events you will see, but they tend to be bystander events.
I am finishing a composite list of all the recurrent TopHat artifacts we call in our compendium of over 450 samples. Most of these are not artifacts of the fusion caller, they are real chimeric reads in usually one of four categories. 1. Read-throughs 2. Tandem duplications 3. Overlapping converging or diverging genes 4.Problems with the reference genome / annotation errors.
When we apply this to most of our TopHat fusion samples >80% of the calls are filtered and we are left with the set of true positives. I am still finishing this compilation up but I will send it to you when I’m done, you can apply it to your TopHat output. We used Ensembl annotations in conjunction with TopHat to generate this. You can call tonight if you have time, we should be home by 10 and are up till 1 usually.
DR
Mar 14, 2014
03/12- Progress Report-小嬿
Fig. 1. CTCF repressed the lytic cycle replications of EBV and KSHV in viral latently infected cells.
> (a). The band of CTCF ectopic-expressed cells is over-exposed.
> (b). The expression level of shCTCF is similar to shLuc.
Fig. 2. Rta binding sites are similar to CTCF binding sites
Fig. 3. 4. EBV Rta expression increased DNA methylation on the target cellular promoter thus interfered with the occupancies of CTCF.
> (a). RNA expression kinetics of cellular genes should be showed in parallel.
> (b). To confirm the methylation states of cellular promoters in EREV8 cells, especially at 24 h of Dox induction.
> (a). The band of CTCF ectopic-expressed cells is over-exposed.
> (b). The expression level of shCTCF is similar to shLuc.
Fig. 2. Rta binding sites are similar to CTCF binding sites
Fig. 3. 4. EBV Rta expression increased DNA methylation on the target cellular promoter thus interfered with the occupancies of CTCF.
> (a). RNA expression kinetics of cellular genes should be showed in parallel.
> (b). To confirm the methylation states of cellular promoters in EREV8 cells, especially at 24 h of Dox induction.
Mar 7, 2014
Biomax tissue microarray informations
Dear老闆:
這是tissue microarray的網址
我挑了一下 (case多及有附normal control的)
Bladder:
BL2081 (203 cases) US. 455
BL802 (80 cases) US. 255
(兩片有60個cases重複)
建議買BL2081
Head and neck, oral cavity:
Old:
OR2081 (98 cases) US.455 (仍有trial等級 US.150)
BC34011 (24 cases) US.140,
HN811 (27 cases) US.215
New:
HN483 (48 cases) US.165 (與OR2081有1個cases重複)
HN803a (80 cases) US.255 (與OR2081有4個cases重複)
建議OR2081 trial等級+ HN803a
給你參考 宛樺 敬上
Mar 6, 2014
Mar 4, 2014
DDR1 manuscript 相關
2015/05/08 (Thu) MS submitted to Research Topic "Oral Oncology"
2015/05/30 (Sat) MS returned, interactive reviews activated:
Editor: Rui Amaral Mendes Catholic University of Portugal; Editor of J Carcinogenesis and Mutagenesis; Adhuant Prof at CWRU
2015/08/28 rejected... by Specialty Chief Editor JeanPascal Machiels
Reviewer#1:
加上morphology圖於Fig 1B
band quantitation
spheroid invasion
Reviewer#2:
移入IHC圖於Fig 5E
補上shRNA sequences and offtarget exp result 於Fig S1B
2015/07/13
Language:
Fine, but there are still numerous errors also in the amended sections.
E.g. Line 356: Interestingly
Line 379: only increment of DDR1 ???
Line 470: cancer
There are more! I just don't have the time to write them down all!
Use of a spell checker and proof-reading by a native English speaker is strongly recommended.
Objective errors:
Response to #1: The standardization of the cell culture medium could confound the experiment. The change of the cell culture medium to DMEM 10% FBS could alter transcriptional programs in cell lines that are normally grown in a different medium. The resulting PTK profiles are therefore flawed.
Response to #2: I still think that the choice of reference cDNA confounds the study. To identify a role in cancer biology, the expression profiles of normal tissue must be compared with that of cancerous tissue. That means a cDNA from normal keratinocytes should have been used as a reference. Comparing a mixed population of expression profiles from unrelated cancer cell lines with the profiles of OSCC cancer cell lines is like comparing apples with pears. The compared cell lines are of different tissue origin and may therefore express DDR1 at completely different levels. These comparisons do therefore not indicate a biological significance of the identified PTKs in oral cancer.
The authors comment below that EGFR expression is even higher in normal human keratinocytes and suggest that this may be caused by continuous EGF supplementation in the medium.
These statements clearly demonstrate that DDR1 could actually be strongly expressed in normal keratinocytes and is therefore not overexpressed in OSCC lines. Furthermore, this study demonstrates the limited applicability of cell line models, especially if grown under different conditions.
Response to #4: The HPV immortalised keratinocyte cell line shows indeed reduced DDR1 levels. However, a normal keratinocyte cell line such as HaCat or HOK is still not shown.
Response to #5: The explanation that DDR1 is the only overexpressed PTK in the analysed OSCC line is based on the assumption that the profiling results are correct. As I pointed out above (Response to #2), these data are flawed.
Response to #6: ANOVA analysis was performed but I'm surprised to see statiscial significance when the means are so close and the error bars overlap.
Introduction:
Lines 110-124: Good attempt but too long. No need to summarise all results.
M&M:
Response to #1: Satisfactory. As mentioned above, a table listing the characteristics of the used cell lines would have been clearer and more appropriate. (Lines 128-142: Content satisfactory. For clarity, a table would have been more suitable.
Response to #2: The technical issues have been sufficiently addressed. However, as outlined above, the profiling data are flawed due to use of an inappropriate control.
Response to 1: The standardization of the cell culture medium could confound the experiment. The change of the cell culture medium to DMEM 10% FBS could alter transcriptional programs in cell lines that are normally grown in a different medium. The resulting PTK profiles are therefore flawed.
I still think that the choice of reference cDNA confounds the study. To identify a role in cancer biology, the expression profiles of normal tissue must be compared with that of cancerous tissue. That means a cDNA from normal keratinocytes should have been used as a reference. Comparing a mixed population of expression profiles from unrelated cancer cell lines with the profiles of OSCC cancer cell lines is like comparing apples with pears. The compared cell lines are of different tissue origin and may therefore express DDR1 at completely different levels. These comparisons do therefore not indicate a biological significance of the identified PTKs in oral cancer.
The new evidence provided by the authors demonstrate that DDR1 could actually be strongly expressed in normal keratinocytes and is therefore not overexpressed in OSCC lines. I can also see no difference between normal controls and OSCC tissue (Fig. 5E). In summary, this study demonstrates the limited applicability of cell line models, especially if grown under different conditions.
Response to 2: The DDR1 levels are lower in the HPV-immortalised cell line but not in DOK. A different control keratinocyte cell line such as OKF6 or HOK should have been included.
Response to 3: has not been addressed by the authors
Response to 10 and 11: The authors do not describe the results of all tested cell lines (only for TW2.6). If there was no effect on cell migration in these cell lines , it should be explained!
Response to 15: Not adressed by the authors.
Response to 17: Unfortunately, the images are worse than the previous version. In the previous version, membranous staining for DDR1 was evident at least in the OSCC sample. What is presented here looks mostly cytoplasmic and there is a very strong background staining. I am concerned about immunostaining of this quality could be scored accurately. The staining pattern has also not been described in the results section.
I still think that the Discussion section lacks substance.
Feb 27, 2014
FGFR fusion related
2/27(四)合作討論會議: BGJ398 A2204 cholangiocarcinoma study
#1由 sufang 在 三, 04/09/2014 - 12:08 發表。
#1由 sufang 在 三, 04/09/2014 - 12:08 發表。
Norvatis人員的報告摘要
1. K650E in FGFR3 mutation is also sensitive to BGJ398.
2. Dan/YiMi的paper是K652E對PD173074無效。
3. TKI258不知道是啥...有點失憶!
Feb 25, 2014
2014-03-04 Journal Club Paper
Dear all:
...20140304 Journal club 輪到我向各位報告。
題目: Hepatocellular Carcinoma-Associated Mesenchymal Stem Cells Promote Hepatocarcinoma Progression: Role of the S100A4-mIR155-SOCS1-MMP9 Axis
Paper 於附檔請查收~~~謝謝大家!!
...20140304 Journal club 輪到我向各位報告。
題目: Hepatocellular Carcinoma-Associated Mesenchymal Stem Cells Promote Hepatocarcinoma Progression: Role of the S100A4-mIR155-SOCS1-MMP9 Axis
Paper 於附檔請查收~~~謝謝大家!!
Feb 24, 2014
QPCR validation of gene fusion on clinical samples
由 sufang 在 二, 04/08/2014 - 09:42 發表 Cholangiocarcinoma Dan Robinson MCTP RNA-seq UMich Yi-Mi
For QPCR validations we use real-time PCR with SYBR green on an ABI instrument such as a StepOne, we set the run for 40 cycles, cDNA from 200 ng or so of toral RNA. Using GAPDH control primers we get a Ct of 18 cycles or so.
For a real fusion, the Ct can be anywhere from 24 to 33 but not much above that. We use ABI Fast SYBR Green mastermix kits with UNG for our PCRs. One trouble with the PCR approach is the number of 3’ partners and the number of possible fusion junctions for each 3’ partner. (The FGFR fusion exon is nearly always the same.)
For QPCR validations we use real-time PCR with SYBR green on an ABI instrument such as a StepOne, we set the run for 40 cycles, cDNA from 200 ng or so of toral RNA. Using GAPDH control primers we get a Ct of 18 cycles or so.
For a real fusion, the Ct can be anywhere from 24 to 33 but not much above that. We use ABI Fast SYBR Green mastermix kits with UNG for our PCRs. One trouble with the PCR approach is the number of 3’ partners and the number of possible fusion junctions for each 3’ partner. (The FGFR fusion exon is nearly always the same.)
For QPCR validations we use real-time PCR with SYBR green on an ABI instrument such as a StepOne, we set the run for 40 cycles, cDNA from 200 ng or so of toral RNA. Using GAPDH control primers we get a Ct of 18 cycles or so.
For a real fusion, the Ct can be anywhere from 24 to 33 but not much above that. We use ABI Fast SYBR Green mastermix kits with UNG for our PCRs. One trouble with the PCR approach is the number of 3’ partners and the number of possible fusion junctions for each 3’ partner. (The FGFR fusion exon is nearly always the same.)
Feb 18, 2014
2014-02-18 Journal Club Paper
Title:Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes by En-Wei
(原文下載)
(原文下載)
Feb 13, 2014
20140212 Lab meeting - ingrid
LabMeeting-Ingrid
由 EVKVLIN 在 三, 02/12/2014 - 00:00 發表 LIN Lab 2016 Ingrid LabMeeting YCK
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