Q RT-PCR

1/19/05

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

 

  1. Precipitating  (If necessary!!! e.g. concentration of RNA < 125ng/uL)

 

    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 (10D=40). 

 

    1. Put 1ug RNA in fresh Eppendorf tube (RNAse free).  Add DEPC ddH20 to make volume up to 40uL. 

 

    1. Add 1/10V 3M Na0Ac pH5.2 (RNAse free) (so 4uL) and 2V 100% EtOH (RNAse free) (so 80 uL).  Mix. 

 

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

 

    1. Pour off supe.  Wash 1x 500 uL RNAse free 70% EtOH.  Spin 5Õ RT.  Pipette of supe, air dry pellet. 

 

  1. DNAse treating

 

    1. If continuing from precipitation step: Resuspend RNA in 8uL DEPC water. 

If  skipped precipitation step: place 1ug of RNA in fresh Eppendorf tube, add DEPC ddH20 to make volume up to 8 uL.  

 

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

 

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

 

  1. cDNA synthesis  (follow Superscript protocol: Invitrogen 11904-018)

 

    1. Mix 8uL of the DNAse treated RNA with:

                                               i.     1uL of random hexamers (50ng/ul)

                                             ii.     1uL 10mM dNTP mix.

 

    1. 65 degrees for 5Õ, on ice for 1Õ.

 

    1. Make master mix with following reagents, then aliquot 9uL/rxn in PCR tubes adding to 10uL RNA/primer mix:

                                               i.     10X RT buffer (2uL/rxn)

                                             ii.     25 mM MgCl2 (4uL)

                                            iii.     0.1M DTT (2uL)

                                            iv.     RNAse (1uL). 

 

    1. 25 degrees for 2Õ

 

    1. Add 1uL superscript II RT to each tube, mix and leave at 25C for 10Õ

 

    1. Using PCR machine:

                                               i.     42C for 50Õ

                                             ii.     70 for 15Õ to terminate reaction

 

    1. Add 1uL RNAse H and incubate for 20Õ at 37C.  Store cDNA at -20