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*[[Hosuk:Lab_Notes|LabNote]] ===Padlock Probe Hybridization Test=== *Continued from [[Hosuk:LabNotes/2013-11-7|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|600px]] ====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 [[Hosuk:LabNotes/2013-11-6|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) *[[Media:Green-Step5_Red-Step6MALAT1_Blue-1stRolonyBlue.zip|Raw]] [[File:Green-Step5_Red-Step6MALAT1_Blue-1stRolonyBlue.png|600px]]
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