E.coli Total RNA Labeling Protocol for Spotted Microarray
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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
| RNA |
12.5ul
|
|
| Random Hexamer |
5mg/ml
|
1ul
|
| Labeling Control (Yeast RNA mix) |
1ul
|
|
| Total |
17ul
|
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~undefined~K~Htd~M~2~1____________~Ktd~M~2~1____________~Kdiv~M10x~K~Hdiv~M~2~1____________~K~Htd~M~2~1____________~Ktd~M~2~1____________~Kdiv~M4ul~K~Hdiv~M~2~1____________~K~Htd~M~2~1________~K~Htr~M~2~1________~Ktr~M~2~1____________~Ktd~M~Ka~M~Ku~MRNA~K~Hu~M_~K~Ha~Msin~K~Htd~M~2~1____________~Ktd~M |
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 ℃ 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
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 500μl). 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 4℃ or -2℃ in dark for further purpose.
Reagents and Suppliers
| Cy3-dUTP | 1mM | Perkinelmer | NEL578 |
| Cy5-dUTP | 1mM | Perkinelmer | NEL579 |
| SuperScript II | 200U/μl | Invitrogen | 18064-014 |
| RNA sin | 20-40U/μl | Promega | N2515 |
| 100 mM dNTP seundefined~K~Htd~M~2~1____________~Ktd~M10X~K~Htd~M~2~1____________~Ktd~MAmersham~K~Htd~M~2~1____________~Ktd~M27~F2035~F01~K~Htd~M~2~1________~K~Htr~M~2~1________~Ktr~M~2~1____________~Ktd~Mpd~AN~B~Ksub~M6_~K~Hsub~MSodium_Salt_~AHexamer~B~K~Htd~M~2~1____________~Ktd~M50U~K~Htd~M~2~1____________~Ktd~MAmersham~K~Htd~M~2~1____________~Ktd~M27~F2166~F01~K~Htd~M~2~1________~K~Htr~M~2~1________~Ktr~M~2~1____________~Ktd~MMicrocon_YM~F30_column~K~Htd~M~2~1____________~Ktd~M | Amicon | 42410 |
