?Note:

Start with 20 mg of total RNA for each labeling reaction.

All solutions that can be filtered should be filtered.

Cy dyes are light sensitive and should ALWAYS be handled in dim light.

RNA Preparation

If RNA is in ethanol, spin down 20 mg of RNA per reaction @ 14000 rpm for 20 min. at 4℃.

Pipette off supernatant and wash pellet with 100ml of 70% ETOH. (prepared with DEPC H2 O)

Spin 5 min. and remove supernatant without disturbing pellet

Air dry pellet 15-20 min at Room Temp (RT). (Caution: if pellet is over dried it is hard to resuspend!)

Resuspend RNA pellet in 12.5 ml DEPC H2O

Heat to RNA to 70 ℃ 5 min, ice 2 min, pulse spin

Labeling

Prepare labeling mix (prepare 1 labeling mix for all smaples labeled at the same time).

1X labeling mix

To the RNA /hexamer mix add 17.0 ml of labeling mix and incubate 10min at RT

Add 1.5 ml of appropriate CyDye dUTP (1mM stock) followed by 1.5 ml SSII reverse transcriptase, mix well by tapping and pulse spin

Incubate 1hr at 42 ℃ in the dark

Add an additional 1.5 ml SSII reverse transcriptase, tap, pulse spin and continue incubation 1hr

Degrade RNA by addition of 2 ml 1N NaOH, vortex, pulse spin and incubate 15 min at 65 ℃

Neutralize by addition of 2 ml 1N HCl, vortex and pulse spin