Hosuk:LabNotes/2013-9-12

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Gene decoding with Barcoded Padlock probes[edit]

Experimental plan[edit]

  • Hybridizing padlock probes on 1st rolonies, and
  • Generate 2nd rolonies over 1st rolonies, and
  • Observe each different fluorescent signal from 2nd rolonies


Procedure[edit]

  • Prepare two barcoded padlock probes - ACTB, RAB7A
Padlock probe for ACTB[edit]
  • ppACTB : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp
    • two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT
    • site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp
    • site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
Padlock probe for RAB7A[edit]
  • ppRAB7A : /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp
    • two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA
    • site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp
    • site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
2nd RCA probe[edit]
  • FISSEQ_ppRCA : GATATCGGGAAGCTGA*A*G --> 18bp


Overall Process[edit]

  1. Prepare each probes – the concentration of each probes are resuspended to 200uM for each ACTB, RAB7A and 2nd RCA primer.
  2. Hybridize Padlock probe – mixture: 1uL ACTB + 1uL RAB7A + 98uL 2x SSC
  3. Add mix, and incubate dish in the oven at 55C for 1hr.
  4. Prepare Ampligase mix - 1uL AmpLigase + 10uL Buffer + 89uL H2O
  5. Aspirate PBS in rolony sample and wash with PBS once
  6. Add AmpLigase mix to sample dish, and incubate in the oven at 45C for 4hr.
  7. Run 2nd RCA from Padlock probes
    • Pre-anneal RCA primer (2uL of 100uM in 98uL 2x SSC) 60°C for 15min
    • Add RCA master mix with aa-dUTP, run RCA for 20 hr.
  8. Fix 2nd RCA with BS(PEG)9, and deactivate BS(PEG)9 with Tris pH8.0 treatment
  9. Image ACTB on 2nd rolonies and Cy5 channel (1st rolonies)
    • Decoding probe mixture: 10uL dcProbe0-Cy3 (10uM)+ 1uL Cy5-adapter for 1st rolony (100uM) + 89uL 2x SSC
    • Pre heat probe mixture at 60C for 5min
    • Add probe mixture to rolony sample and cool down at RT for 10min
    • Wash with PBS twice and add 2mL PBS, and imaging
  10. Image RAB7A on 2nd rolonies and Cy5 channel (1st rolonies)
    • Strip probes by adding 80% Formamide in 2x SSC (pre-heat at 60C for 5min, and add and incubate at RT for 10min, and wash with PBS)
    • Decoding probe mixture: 10uL dcProbe1-Cy3 (10uM)+ 1uL Cy5-adapter for 1st rolony (100uM) + 89uL 2x SSC
    • Pre heat probe mixture at 60C for 5min
    • Add probe mixture to rolony sample and cool down at RT for 10min
    • Wash with PBS twice and add 2mL PBS, and imaging


  • 2nd Rolony generation at 09/10~09/11, imaging at 09/11


Result for 1st try[edit]

result[edit]

  • 1st Padlock probes for ACTB were not seen well, too sparse and too few
  • 2nd Padlock probes for RAB7A were many, more than ACTB.
  • Currently we don't know why there are not many signal on 2nd rolonies, becuase of...
    • Ampligase reaction were not good? or low efficiency? becuase of temperature in oven? incubation time?
    • Not enough padlock probes? or poor hybrizing efficiency?
  • need to figure out
  • We need to check all of 2nd rolonies, so we are going to use FITC-labeled probes which is the sequence for the region of 2nd RCA probes (488nm dye labeled CTTCAGCTTCCCGATATC)


  • We didn't do quantitative analysis yet, and will image this sample (RAB7A) with Confocal.


Number of Rolonies[edit]

  • Use Matlab code (D:\My Documents\..\04. Matlab\Rolony_Counting_3.m)
  • Use epi FL ficture (2013-09-11)
  • ACTB : 1st Rolony --> 3931, 2nd(ACTB) --> 67
    • ACTB/1st = 1.70%
  • RAB7A : 1st Rolony --> 4085, 2nd(RAB7A) --> 204
    • RAB7A/1st = 4.99%


Figures - epi FL[edit]


ACTB on 2nd rolonies[edit]
1st Rolonies, Cy5 (Raw) ACTB on 2nd Rolonies, Cy3 (Raw) Mergerd
File:S062319 1stRolony-Cy5 GeneACTB-Cy3 P02 Fig01 FLCy5 red.jpg File:S062319 1stRolony-Cy5 GeneACTB-Cy3 P02 Fig02 FLCy3 cyan.jpg File:Composite ACTB P02.jpg


RAB7A on 2nd rolonies[edit]
1st Rolonies, Cy5 (Raw) RAB7A on 2nd Rolonies, Cy3 (Raw) Mergerd
File:S062319 1stRolony-Cy5 GeneRAB7A-Cy3 P02 Fig02 FLCy5 EM20 red.jpg File:S062319 1stRolony-Cy5 GeneRAB7A-Cy3 P02 Fig01 FLCy3 EM40 cyan.jpg File:Composite RAB7A P02.jpg


Figures - Confocal[edit]

  • Image RAB7A sample only
  • 20x obj, oil
  • Red : Cy5 (1st Rolony)
  • Green : Cy3 (2nd, RAB7A)
  • 2048 x 2048 resolution, FOV : 580um x 580um with zoom 1x
  • Z stack(10um), and maximum projection


location 1, Zoom = 1x (Raw, Cropped) location 2, Zoom = 2x (Raw)
File:Confocal RAB7A Red-1stRolony Green-2ndRolony pos01 zoom1x 2.png File:Confocal RAB7A Red-1stRolony Green-2ndRolony pos02 zoom4x.png


Counting RAB7A (added at 2013-10-25)[edit]
  • Rolony_Counting_10.m
  • 1st Rolony : 29641
  • 2nd Rolony (RAB7A) : 2811
  • Overlapped RAB7A : 791
    • 791/29641 = 2.67%
    • Raw

File:RAB7A Overlapped 1stRolony 2013-09-12.png