About Us > Facilities > Ginsberg Protocol

    For LCM captured cells:

  • Thaw samples (LCM cap in Trizol) on ice
  • Move Trizol/cell solution into 1.5 ml microtube and add 100 μl of Chloroform (Sigma C- 2432)
  • Shake vigorously for 15 sec and let sit at RT 1-2 min
  • Spin down at 12,000g for 5–10 min (old protocol 15 min) @ 4º C
  • Aspirate SN to fresh tube and add 250 μl of isopropanol (Sigma  I-9516) and 2 μl of Linear Acrylamide (Ambion 9520 5mg/ml)
  • Mix and spin @ 12,000g for 20 min @ 4º C
  • Rinse pellet with 80% EtOH and air dry (not more than 5 min) inverted and resuspend pellet in 5.5 μl of ddH2O (Rnase free)

    RNA Amplification protocol:

  • Transfer samples to 0.5ml thin wall PCR tubes (usually volume is 6 μl, otherwise adjust to 6 μl) and add:
    • 1 μl Poly T primer (100 ng/ μl) (primer sequence TTT TTT TTT TTT TTT TTT TT)
  • Mix samples and incubate in  PCR machine: 65 ºC for 2 min, then 45º C for 1 min
  • While samples in PCR machine, heat denature TC primer (5T7C 100 ng/ μl) to 70º C for 1-2 min and put on ice.(primer sequence:    AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC GAG AGG CCC)
  • Set up 1st strand amplification, add these ingredients to rxn above (can make master mix):

    • 5X First strand buffer -- 4 μl per reaction
    • 0.1M DTT -- 1 μl per reaction
    • 10mM dNTPs (A,T,C,G) -- 1 μl per reaction
    • First strand enzyme mix -- 2 μl per reaction

    • (Superscript III {Invitrogen #50575 200U/ul and SuperRNase Inhibitor Ambion #2696  20 U/ul 1:1 ratio)
    • TC primer (5T7C 100 ng/μl)-- 1 μl per reaction
    • ddH2O to 20μl reaction total volume
  • Incubate reaction at 50 ºC 60 min {if using SSII, lower temp to 42 ºC}, then 65 ºC 15 min

  • Set up “2nd strand synthesis rxn” (don’t actually do 2nd strand synthesis), add to reaction above:
    • 0X taq PCR buffer -- 10 μl per reaction
    • RNase H (Ambion 2293 10 U/ul) -- 1 μl per reaction
    • ddH2O -- 69 μl per reaction (100 μl reaction volume total)

  • Incubate reaction 37 ºC 30 min, then 95 ºC 3 min, then 60 ºC 3 min
  • **All above reactions done in PCR machine

  • Use Sartorius columns vivaspin 500 10kD MW spin column (VS0101) down to 13 μl, adjust volume if below 13 μl with ddH2O
  • Set up T7 Transcription reaction for 40 μl total reaction volume:
    • cDNA -- 13 μl
    • 5X Transcription buffer (Epicentre BP1001) -- 8 μl
    • 2.5 mM NTPs (A,G,C) -- 2 μl
    • 100 μM UTP (cold) -- 1 μl
    • 0.1M DTT -- 1 μl
    • SuperRNase Inhibitor -- 1 μl
    • go behind shield then add:/li>
    • 33P-UTP  (PE:  NEG307H001MC) -- 12 μl/li>
    • T7 RNA polymerase (Epicentre TH950K 1KU/ul) -- 2 μl
  • Mix up and incubate at least 4 hrs at 37 ºC (max of 6 hrs incubation)
  • While incubating T7 Transcription reaction, mix up pre-hybridization buffer
    • 100% Formamide (Fisher) -- 10 ml/rxn
    • 50X Denhart’s solution (AmResCo) -- 2 ml/rxn
    • 20X SSPE (Ambion 9767) -- 6 ml/rxn
    • 10% SDS (Ambion 9823) -- 200 μl /rxn
    • MilliQ H2O -- 1.4ml/rxn
    • Salmon Sperm DNA (Eppendorf 955155629) -- 400μl /rxn

    (denatured 84* 4 min, 200 ug/ml)
  • Add 20 ml to each blot in hybridization tubes (wrap blot in mesh, label tube with blot # and date of blot and reaction)
  • Incubate at 42 ºC for 4 hrs on rotator(until T7 reaction done)
  • Add T7 reaction to correct tubes and incubate on rotator o/n at 42 ºC

  • Next day: 16-18 hr incubation
  • Discard hybridization solution behind shield in bottle
  • Wash blots with 20 ml of 2X SSC buffer (2X SSC {Ambion9763} + 0.1% SDS) for 15 minutes at 37*C on rotator
  • Discard buffer behind shield
  • Wash blots with 20 ml of 1X SSC buffer (1X SSC + 0.1% SDS) for 15 minutes at 37*C on rotator
  • Discard buffer behind shield
  • Wash blots with 20 ml of 0.5X SSC buffer (0.5X SSC + 0.1% SDS) for 15 minutes at 37*C on rotator
  • After done, put blots between 2 pieces of plastic wrap and expose to phosphoscreens
  • Incubate mesh, caps and bottles with MilliQ H2O and add 200 μl of 10% SDS o/n (wash 3 times total)

Next day: 24-30 hr exposure develop phosphoscreens using Storm 840 phosphoimager, save each image and go onto image analysis