Hosuk:LabNotes/2014-8-10

From ZhangLabWiki
Jump to navigation Jump to search

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