E.coli Total RNA Labeling Protocol for Spotted Microarray

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^oC.
• Pipette off supernatant and wash pellet with 100ml of 70% ETOH. (prepared with DEPC H₂O)
• 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

RNA 12.5ul Random Hexamer 5mg/ml 1ul Labeling Control (Yeast RNA mix) 1ul

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Total 17ul

Heat to RNA to 70 ^oC 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

First Strand Buffer 5x 8ul DTT 0.1M 4ul dNTPs(low dTTP)** 10x 4ul RNAsin 1ul

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Total 17ul

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

Clean up Labeled Probes

• Prewash Microcon-30 microfilter by adding 450ml miliQH