Q RT-PCR

4/19/06

 

Part 2: cDNA preparation (precipitate, DNAse, cDNA synthesis)

 

1.     Spec RNA (2uL in 100). Make sure to use filter tips and try not to contaminate with RNAses. Keep RNA on ice. Make sure to change the program so that it reads RNA (1OD=40).

 

  1. Put 1ug RNA in fresh Eppendorf tube, RNAse free. Add DEPC ddH2O to make volume up to 40uL.

 

  1. Add 1/10V 3M NaOAc pH5.2  (RNAse free) (so 4uL) and 2V 100% EtOH (RNAse free) (so 80uL). Mix.

 

  1. Keep 20Õ at –80C (bury tube in the frost) and spin 20Õ at 4C.

 

  1. Pour off supe. Wash 1x 500uL RNAse free 70% EtOH. Spin 5Õ RT.  Pipette off supe.

 

  1. Air dry pellet. Resuspend RNA in 8uL DEPC water. Now ready to DNAse treat.

 

  1. ALTERNATIVELY: if 1ug DNA is in less than 8uL can just take 1ug DNA, make to 8uL with DEPC and proceed with step 8.

 

  1. Add 1uL DNAse reaction buffer and 1uL DNAse (from ÒQ-RT-PCRÓ box in freezer). Mix carefully, and leave 15Õ at RT.

 

  1. Add 1uL 25uM EDTA (from QRT-PCR box). Mix carefully, and place at 65 degrees for 10Õ to kill DNAse.  Spin down tube and proceed to cDNA synthesis.

 

  1. Follow Superscript II protocol (Invitrogen 11904-018).

 

  1. Mix 8uL of DNAse sample with with 1uL random hexamers (50ng/ul) or oligo dT primer and 1uL 10mM dNTP mix. I do this in PCR tubes and do all subsequent steps in the PCR machine, using the ÒinstantÓ function and an RT program. Label the tubes with numbers for the samples on the side and the date on the top. Record the contents of the tubes.

 

  1. 65C for 5Õ, on ice 1Õ.

 

  1.  Make master mix 10X RT buffer (2uL/rxn),  25mM MgCl2 (4uL) 0.1M DTT (2uL/rxn), RNAse out (1uL). Aliquot 9uL/rxn in PCR tubes adding to 10uL RNA/primer mix.

 

  1. 25C for 2Õ.

 

  1. Add 1uL Superscript to each tube. Vortex briefly to mix and return to PCR machine.

 

  1. 42C for 50Õ.

 

  1. 70C for 15Õ to terminate reaction.

 

  1. Add 1uL RNAseH and incubate for 20Õ at 37C. Store cDNA at –20C.