For LCM captured cells:

  • Thaw samples (LCM cap in Trizol) on ice
  • Move Trizol/cell solution into 1.5 ml microtube and add 100 ml 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 ml of isopropanol (Sigma  I-9516) and 2 ml 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 ml of ddH2O (Rnase free)

RNA Amplification protocol:

  • Transfer samples to 0.5ml thin wall PCR tubes (usually volume is 6 ml, otherwise adjust to 6 ml) and add:
    • 1 ml Poly T primer (100 ng/ ml) (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/ ml) 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 ml per reaction
    • 0.1M DTT                                           1 ml per reaction
    • 10mM dNTPs (A,U,C,G)                  1 ml per reaction
    • First strand enzyme mix                   2 ml per reaction

(Superscript III {Invitrogen #50575 200U/ul and SuperRNase Inhibitor Ambion #2696  20 U/ul 1:1 ratio)

    • TC primer (5T7C 100 ng/ml)            1 ml per reaction
    • ddH2O to 20ml 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:
    • 10X taq PCR buffer                                      10 ml per reaction
    • RNase H (Ambion 2293 10 U/ul)                1 ml per reaction
    • ddH2O                                                                        69 ml per reaction (100 ml 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 Millipore montage PCR columns purify samples and get rid of unbound primers (see inset of columns for protocol:  Cat # UFC7PCR50)

  • Once reactions purified by column, measure volume and adjust (by speed vac) to 19 ml (FOR RADIOACTIVE LABELING) OR measure volume and adjust (by speed vac) to 10 ml (FOR BIOTINYLATED/FLUORESCENT LABELING) 

LABELING CAN PROCEED AT THIS POINT. WE PROVIDE PROTOCOLS FOR RADIOACTIVE LABELING AND BIOTINYLATED LABELING
  • RADIOACTIVE LABELING
  • Set up T7 Transcription reaction for 40 ml total reaction volume:
    • cDNA                                                                                      19 ml
    • 5X Transcription buffer (Epicentre BP1001)                     8 ml
    • 2.5 mM dNTPs (A,G,C)                                                        2 ml
    • 100 mM dUTP (cold)                                                             1 ml
    • 0.1M DTT                                                                               1 ml
    • SuperRNase Inhibitor                                                           1 ml
    • go behind shield then add:
    • 33P-UTP         (GE Healthcare BF-1002-1MCI)                 6 ml
    • T7 RNA polymerase (Epicentre TH950K 1KU/ul) 2 ml
  • 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 ml /rxn
    • MilliQ H2O                                                                              1.4ml/rxn
    • Salmon Sperm DNA (Eppendorf 955155629)                  400ml /rxn

(denatured 84* 4 min, 200ng/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 ml 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

IVT for TC RNA Amplification: Biotinylated/Fluorescent Probe Labeling

1.         Prepare IVT Master Mix (prepare enough mix for the total number of samples plus two extra). Calculate volumes of the mix for x # of samples + control + one. This protocol is based upon using the BioArray RNA transcript labeling kit (Enzo), although any biotinylated and/or fluorescent labeling protocol is suitable (minor modifications may apply).

2.         IVT Master Mix consists of (on wet ice):

10X Hybridization reaction buffer                                              4 µl

10X Biotin labeled ribonucleotides                                            4 µl

            10X DTT                                                                                 4 µl

10X RNase inhibitor mix                                                           4 µl

            18.2 mega Ohm RNase-free water                                           12 µl

20X T7 RNA polymerase                                                         2 µl

(or T7 RNA polymerase from Epicentre; Appendix III  2 µl)

3.         The IVT Master Mix should comprise of 30 µl per reaction.

4.         Add 10 µl of double stranded TC sample to the IVT Master Mix (final volume of the reaction is 40 µl).

5.         Mix thoroughly (very important!!). Quick spin and incubate for 5 h at 37 °C.

6.         TC RNA amplified products are now ready for purification (if desired), fragmentation (if desired), and application to cDNA or oligonucleotide arrays. There are numerous published protocols for hybridization of labeled probes to microarray platforms, subsequent washing, and imaging protocols (e.g., www.affymetrix.com, www.enzo.com).