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LNA26/11.2006
 
 
LNA™ for in situ detection of mRNA in fixed cells

 
Method prepared by R. W. Dirks, Leiden University Medical Center, Leiden, The Netherlands.

Rat9G cells are grown on microscope slides and immediate early HCMV mRNA expression is induced (in approximately 30% of the cells) by incubating cells with cycloheximide for 4-6 hours. After this:

- Wash cells shortly with PBS;
- Fix cells in 4% formaldehyde/5% acetic acid in PBS for 15 min;
- Wash cells 2 x 5 min with PBS;
- Treat cells with pepsin (0.1% in 10 mM HCl) for 1 min at 37ºC;
- Wash cells 2 times shortly with water;
- Dehydrate through 70%-, 90%- and 100% ethanol;
- Air dry.
- Put 10 μl of diluted probe (see below) on a slide and cover with a 18x18 mm

cover slip;

- Denature probe + slide at 80ºC for 75 sec;
- Hybridize for 30 min at 37ºC in a moist chamber;
- Wash off the cover slip by immersion in 2xSSC, 0.1% Tween-20;
- Wash 3 x 5 min with 0.1xSSC at 65ºC;
- Dehydrate through 70%-, 90%- and 100% ethanol;
- Air dry;
- Bring on 20 μl of DAPI + antifade and cover with a 24x50 mm coverslip.
 
Probe dilution:
The LNA™ mRNA detection probe is diluted with H2O to a concentration of 100 nM. To this solution is added 50 μl deionized formamide + 10 μl 20xSSC + 20 μl 50% dextran sulphate + 10 μl 0.5 M Sodium phosphate pH 7 to give a final concentration of the LNA™ mRNA detection probe of 10 nM.

The above probe solution is diluted 50x in 50% deionized formamide, 2xSSC, 50 mM Sodium phosphate pH 7, 10% dextran sulphate, and used for hybridization..  


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