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RNA Labeling Protocol

 

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 4oC.
  • 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
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
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 miliQ H2O and spinning for 10 min. @ 12,000 RPM.
  • Add 450ml miliQ H2O to each of the probe samples (or total 500ul). Mix thoroughly by pipetting up and down. Transfer samples to separate Microcon-30 microfilters. (Amicon)
  • Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.
  • Add 450 ml miliQ H2O to the probe and gently mix by pipetting up and down. Be careful not to touch the filter at the bottom of the filtration unit.
  • Spin 10 minutes at 12,000 RPM
  • Repeat step 4 , spin 12min to get smaller volume.
  • Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.
  • Transfer recovered probe to the appropriate partner probe.
Note: Probes can be combined after the first wash step.
Probe can be stored at 4C or -20C in dark for further purpose.

 

Reagents and Suppliers

Cy3-dUTP 1mM Perkinelmer NEL578
Cy5-dUTP 1mM Perkinelmer NEL579
SuperScript II 200U/ul Invitrogen 18064-014
RNAsin 20-40U/ul Promega N2515
100 mM dNTP set** 10X Amersham 27-2035-01
pd(N)6 Sodium Salt (Hexamer) 50U Amersham 27-2166-01
Microcon YM-30 column Amicon 42410

Other reagents: 20X SSC, TE pH7.4, 10% SDS, 500 mM EDTA, 1M NaOH, 1M Tris-HCl pH7.5, sterile dH2O and DEPC H2O
* comes lyophilized, must be resuspended at specified concentration.

**for 10X stock: 5 mM each of dA, dG, dC and 2 mM of dT in DEPC H2O

 
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