Hosuk:LabNotes/2014-7-4

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NCIH-1975 with T48 RT Primer[edit]

  • Matt's RT Primers (48 primers) for hybridizing only mRNA (hopefully)
  • S1 : T48 RT primer, S2 : rHexamer


Procedure[edit]

  • 07/02
    1. Fix NCIH1975 by adding 10% formalin incubating at RT for 15min, wash with PBS three times
    2. Add 0.25% Triton X-100 in DEPC-PBS and incubate for 10min, wash with PBS three times
    3. Add 0.1N HCl and incubate 1min, wash with PBS three times
    4. Wash with 2 ml PBS three times
    5. Add RT mix (Hexamer, -a-dUTP)
      • DEPC-H2O 161uL
      • M-MuLV Buffer 20uL
      • 25mM dNTP 2uL
      • 100uM RT Primer 5uL
      • Rnase Inhibitor 2uL
      • M-MuLV reverse transcriptase 10 uL
    6. Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for 12 hr.


  • 07/03
    1. Wash with PBS once, skip BS(PEG)9
    2. Add RNase H mix, incubate for 1hr. at 37C
      • RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
    3. Wash with nucleaus-free H2O twice
    4. Add CircLigase II mix, incubate at 60C for 2.5hr.
      • H2O 128uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 40uL
      • CircLigaseII 2uL
    5. RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr. --> 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    6. 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    7. Aspirate and add 2XSSC, incubate at 60C for 10min
    8. Aspirate and Wash with 2X SSC, 1X SSC and PBS once each
    9. Prepare RCA reaction mix
      • H2O 172uL
      • Buffer 20uL
      • 25mM dNTP 2uL
      • 2mM a-dUTP 4uL
      • Phi29 2uL
    10. Add RCA mix, incubate at 30C for 19.5hr


  • 07/04
    1. Wash with PBS once, Prepare 4uL of BS(PEG)9 in 196uL PBS, add 200uL BS(PEG)9 mix, incubate for 1hr at RT
    2. Wash with PBS once, add Tris pH 8.0, incubate for 30min
    3. Wash with PBS once
    4. Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
    5. Preheat detection Probe at 80C for 5min
    6. Add to sample and incubate at RT for 10min
    7. Wash two times for 1 minute each with 1 mL 1X PBS
    8. Imaging


Result[edit]

  • Both samples have most of rolonies inside nuclear, so much localized…
    • it cased maybe fixation condition or cell culture condition.
  • Random hexamer sample has more rolonies than T48 RT primer has.


  • Counts (S1:T48, S2 Random Hexamer)

File:NCIH1975 Count Table S1.jpg File:NCIH1975 Count Table S2.jpg


File:NCIH1975 S1 S2 20x.jpg File:NCIH1975 S1 S2 63x.jpg File:NCIH1975 S1 S2 63x Zoom4.jpg