Hosuk:LabNotes/2014-8-10: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Hosuki78
(Created page with "*LabNote ===Cancer Mutation detection=== ====Sample==== *Sample **NCIH1975_S1_2014-08-05 : -dUTP, rHex  45C **NCIH1975_S2_2014-08-03 : +dUTP, rHex  ...")
 
>Hosuki78
No edit summary
 
(5 intermediate revisions by the same user not shown)
Line 2: Line 2:


===Cancer Mutation detection===
===Cancer Mutation detection===
====Sample====
====Decoding, and Analyzing====
*Sample
*Images
**NCIH1975_S1_2014-08-05 : -dUTP, rHex  45C
**ch00 - dcProbe[#]-488
**NCIH1975_S2_2014-08-03 : +dUTP, rHex  60C --> will use this later!
**ch01 - dcProbe[#]-Cy3
**ch02 - Bright field


*Steps
**Step1 : dcProbe0-488
**Step2 : dcProbe0-Cy3
**Step3 : dcProbe1-488
**Step4 : dcProbe1-Cy3
**...
**Step9 : dcProbe4-488
**Step10 : dcProbe4-Cy3


====Run Ampligase + Klentaq and 2nd RCA====
*Reaction Mix
[[File:Ampligase_Reagent_2014-08-07.jpg|600px]]


*I've taken images from 3 different positions (Pos1, Pos2, and Pos3)
**However the result that successfully analyzed was from only Pos1


*Start Ampligase+Klentaq : 08/05, 3:30pm (Just use NCIH1975_S1_2014-08-05), ~21.5hr at 45C
*Start RCA : 08/06, 2:40pm
**1uM ppRCA in 200uL 2x SSC, add and incubate at 42C for 36min,
**Wash with 2x SSC twice, 1x SSC twice, 1x PBS twice, then add Phi29 mix
*Finish : 08/07 (RCA for  ~18.5hr)




====Decoding====
*Procedure
*Decoding – 08/07
*#Figure out the offset pixels with BF image
*Sample : NCIH1975_S1_2014-08-05 : -dUTP, rHex
*#*Pos1
*1st Rolony – Cy3 : 1uM
*#*Standard : Step3
*dcProbe0~4 : 488, Cy3 : 1uM
*#*Step1 : X = 1, Y = -25
**dcProbe[#]-488 and dcProbe[#]-Cy3 are mixed in a sampe 100uL tube
*#*Step2 : X = 0, Y = -8
*#*Step3 : X = 0, Y = 0
*#*Step4 : X = -8, Y = 2
*#*Step5 : X = -8, Y = 9
*#Make maximum intensity projection(MIP) images at each step
*#Shift and crop the MIP images of each step : Crop size became 26 based on the offset max. of pixel
*#Run Script (RolonyDataInfo_v9a.m and Rolony_Decoding_v14b.m)
*#Make one MIP image with the MIP image of each step : for a mask image




*Step
====Result====
*#Add dcProbe[#], incubate for 10min, image
*Too few signal was decoded, it maybe causes from very low number of targets, either from few 1st rolony left while Padlock probe capture or originally few targets in the cells … not sure though.
*#Strip with 80% formamide(pre-heated at 70C) for 15min, wash with PBS twice
*For the future, Need to try ampligase+Klentaq reaction temp. at 60C
*#Repeat applying dcProbe and imaging




*Result : under anaylzinging
*Decoding condition : Search Radious = +/-1.5 px
*Red : BRAF-MU, Green : BRAF-WT, Blue : T790M-MU, Cyan : T790M-WT,  Gray : all signals (masked, and maximum projected signals)
[[File:CompositeImg_DecodeResult1_NCIH1975_2014-08-05.jpg|800px]]
[[File:DecodeResult1_NCIH1975_2014-08-05.jpg|200px]]
 
 
====Images====
*Decoding condition : Search Radious = +/-3 px
*Red : BRAF-MU, Green : BRAF-WT, Blue : T790M-MU, Cyan : T790M-WT,  Gray : all signals (masked, and maximum projected signals)
[[File:CompositeImg_DecodeResult2_NCIH1975_2014-08-05.jpg|800px]]
[[File:DecodeResult2_NCIH1975_2014-08-05.jpg|200px]]

Latest revision as of 23:23, 12 August 2014

Cancer Mutation detection[edit]

Decoding, and Analyzing[edit]

  • Images
    • ch00 - dcProbe[#]-488
    • ch01 - dcProbe[#]-Cy3
    • ch02 - Bright field
  • Steps
    • Step1 : dcProbe0-488
    • Step2 : dcProbe0-Cy3
    • Step3 : dcProbe1-488
    • Step4 : dcProbe1-Cy3
    • ...
    • Step9 : dcProbe4-488
    • Step10 : dcProbe4-Cy3


  • I've taken images from 3 different positions (Pos1, Pos2, and Pos3)
    • However the result that successfully analyzed was from only Pos1


  • Procedure
    1. Figure out the offset pixels with BF image
      • Pos1
      • Standard : Step3
      • Step1 : X = 1, Y = -25
      • Step2 : X = 0, Y = -8
      • Step3 : X = 0, Y = 0
      • Step4 : X = -8, Y = 2
      • Step5 : X = -8, Y = 9
    2. Make maximum intensity projection(MIP) images at each step
    3. Shift and crop the MIP images of each step : Crop size became 26 based on the offset max. of pixel
    4. Run Script (RolonyDataInfo_v9a.m and Rolony_Decoding_v14b.m)
    5. Make one MIP image with the MIP image of each step : for a mask image


Result[edit]

  • Too few signal was decoded, it maybe causes from very low number of targets, either from few 1st rolony left while Padlock probe capture or originally few targets in the cells … not sure though.
  • For the future, Need to try ampligase+Klentaq reaction temp. at 60C


  • Decoding condition : Search Radious = +/-1.5 px
  • Red : BRAF-MU, Green : BRAF-WT, Blue : T790M-MU, Cyan : T790M-WT, Gray : all signals (masked, and maximum projected signals)

File:CompositeImg DecodeResult1 NCIH1975 2014-08-05.jpg File:DecodeResult1 NCIH1975 2014-08-05.jpg


Images[edit]

  • Decoding condition : Search Radious = +/-3 px
  • Red : BRAF-MU, Green : BRAF-WT, Blue : T790M-MU, Cyan : T790M-WT, Gray : all signals (masked, and maximum projected signals)

File:CompositeImg DecodeResult2 NCIH1975 2014-08-05.jpg File:DecodeResult2 NCIH1975 2014-08-05.jpg