Hosuk:LabNotes/2013-11-8
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Padlock Probe Hybridization Test
- Continued from 11/7
Aligning
- 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
Description of each Steps
- 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
- All 3 padlock probes in Step3 --> 5326
- MALAT1 padlock probes in Step5 --> 5022
- Overlapped with Step5 and Step3 --> 4498
- 1st Rolony (Step6): 6266
- MALAT1 (from Step6): 1577 --> Overlapped : 1043 --> Somehow the number of MALAT1 was significantly dropped after adding Cy5 labeled 1st Rolony detection probe, I think it may cause from FRET???
- 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.
- MALAT1 in Step3 (3 padlock probes (ppACTB, ppRAB7A, ppMATAL1) annealed : 1238
- MALAT1 in Step5 (1 padlock probe (ppMATAL1) annealed : 1183
- MALAT1 genes on 1st Rolony were detected much more than RAB7A or ACTB
- (ACTB+RAB7A+MALAT1) / 1st Rolony = 1238/6266 = 19.76%
- MALAT1 / 1st Rolony = 1183/6266 = 18.88%
- The overlapped number of MALAT1 from Step6 is 1043, and overlapped number of MALAT1 from Step5 is 1183, but the counted number of MALAT1 in Step5 BEFORE overlapped to 1st Rolony was 5022 --> Then the real number of MALAT1 would be about 1043~1183, and the most of signals in Step3 or Step5 were not real from rolony...
- Green : MALAT1 in Step5 (Count = 5022, Overlapped Count = 1183)
- Red : MALAT1 in Step6 (Count = 1577, Overlapped Count = 1043)
- Blue : 1st Rolony in Step6 (Count = 6266)
- Raw