Hosuk:LabNotes/2013-11-8: Difference between revisions
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*All 3 padlock probes in Step3 --> 5326 | *All 3 padlock probes in Step3 --> 5326 | ||
*MALAT1 padlock probes in Step5 --> 5022 | *MALAT1 padlock probes in Step5 --> 5022 | ||
*Overlapped with Step5 and Step3 --> 4498 | **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 ) | *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. | **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 [[Hosuk:LabNotes/2013-11-6|11/6]]''' | |||
*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 = | *Green : MALAT1 in Step5 (Count = 5022, Overlapped Count = 3700) | ||
*Red : MALAT1 in Step6 (Count = 1577, Overlapped Count = | *Red : MALAT1 in Step6 (Count = 1577, Overlapped Count = 1047) | ||
*Blue : 1st Rolony in Step6 (Count = 6266) | *Blue : 1st Rolony in Step6 (Count = 6266) | ||
*[[Media:Green-Step5_Red-Step6MALAT1_Blue-1stRolonyBlue.zip|Raw]] | *[[Media:Green-Step5_Red-Step6MALAT1_Blue-1stRolonyBlue.zip|Raw]] | ||
[[File:Green-Step5_Red-Step6MALAT1_Blue-1stRolonyBlue.png|600px]] | [[File:Green-Step5_Red-Step6MALAT1_Blue-1stRolonyBlue.png|600px]] |
Revision as of 19:57, 8 November 2013
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
- 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
- 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