Hosuk:LabNotes/2013-11-8
Jump to navigation
Jump to search
Padlock Probe Hybridization Test[edit]
- Continued from 11/7
Aligning[edit]
- I touched dish while moving the stage adapter back between Step3, and Step5
- Step3 : Add 3 padlock probes together (ppRAB7A + ppACTB + ppMALAT1), and image the signal from all padlock probes by using 'Shifted FISSEQ_2ndRCAprimerDye' (universal seq. for all padlock probes)
- Step5 : Add only ppMALAT1 padlock probe, and detect signal by 'Shifted FISSEQ_2ndRCAprimerDye'
- So I need to look for the same spot, and found it.
- Image of Step 5 was shifted (Visual observation by ImageJ) --> 8 px to left, 4 px to up
- Step5 Image was aligned by GlobalAlign.m
- However, x_offset was needed to be tuned, by 1 px, so I added x_offset = x_offset+1, and do SHIFTED_IMG = circshift(IMAGE, [y_offset x_offset]) for final aligned image
- Green : Step3, Red : Step5
File:Alighed Step3Green Step5Red.png
MALAT1, 1st Rolony counting[edit]
Description of each Steps[edit]
- Step3 : Add 3 padlock probes together (ppRAB7A + ppACTB + ppMALAT1), and image the signal from all padlock probes by using 'Shifted FISSEQ_2ndRCAprimerDye' (universal seq. for all padlock probes)
- Step5 : Add only ppMALAT1 padlock probe, and detect signal by 'Shifted FISSEQ_2ndRCAprimerDye'
- Step 6 : add Cy5 labeled 1st Rolony probe WITHOUT stripping from step5
- PISA setting
- Gaussian Std : 4, area upper: 100, area lower: 2, axratio lower: .7, circ upper: 1.5, circ lower: 0.8, perim conn: 8, bkgmult lower: 9
Count[edit]
- All 3 padlock probes in Step3 --> 5326
- MALAT1 padlock probes in Step5 --> 5022
- Overlapped with Step5 and Step3 --> 4498
- I counted Rolony after aligning Step6 images based on Step3 image (8 px to right, 4 px to down )
- Since 1st Rolony image is in Step6, I counted MALAT1 rolony in Step3 and Step5 overlapped to aligned 1st Rolony in Step6, after aligning Step6 image.
- 1st Rolony (Step6): 6266
- MALAT1 in Step3 (3 padlock probes (ppACTB, ppRAB7A, ppMATAL1) annealed : 4094 --> (ACTB+RAB7A+MALAT1) / 1st Rolony = 4094/6266 = 65.34%
- MALAT1 in Step5 (1 padlock probe (ppMATAL1) annealed : 3700 --> MALAT1 / 1st Rolony = 3700/6266 = 59.05%
- MALAT1 genes on 1st Rolony were detected much more than RAB7A or ACTB.
- (ACTB+RAB7A) would be (4094-3700) = 394, which is roughly similar to the counting of the previous result 11/6, of course the image spot of 11/7 is different from of 11/6 but the range of the number of (ACTB+RAB7A) is similar to both result of 11/6 and 11/7.
- MALAT1 (from Step6): 1577 --> Overlapped : 1047 --> Somehow the number of MALAT1 was significantly dropped after adding Cy5 labeled 1st Rolony detection probe, I think it may cause from FRET???
- Green : MALAT1 in Step5 (Count = 5022, Overlapped Count = 3700)
- Red : MALAT1 in Step6 (Count = 1577, Overlapped Count = 1047)
- Blue : 1st Rolony in Step6 (Count = 6266)
- Raw