RIBONUCLEASE PROTECTION ASSAY (RPA) is a method to simultaneously quantify multiple mRNAs in a total RNA extract.  This method utilized ³²P-labeled RNA probes that hybridize to their cognate mRNA creating a region of double-stranded RNA that is resistant to RNase digestion.  These dsRNAs are resolved on a sequencing gel and visualized and quantitated by phosphorimager.  The user can select from a range of multiple-probe template sets (cytokines, apotosis genes, stress response genes, DNA repair genes etc.) available from PharMingen (www.pharmingen.com).

The overall procedure includes:
1. Total RNA extraction from the sample
2. Radio-labeling of the probe set
3. Hybridization of probes with RNA followed by RNase digestion.
4. Gel electrophoresis of the hybridized and RNase-digested mRNA and visualization/quantitation by phosphorimages

 
Charges:

RNA preparation and RPA analysis (1 gel - 20 samples) for OMRF investigators - $200
RNA preparation and RPA analysis (1 gel - 20 samples for OUHSC investigators - $400
(These charges do not include the cost of probe sets or ³²P radioisotope)
 

RNA INTERFERENCE (RNAi) is a recently discovered biological phenomenon that has been adapted to provide a tool for studying gene expression.  By transfecting small double-stranded fragments of RNA of known sequence into cells, specific mRNAs can be targeted for degradation resulting in knockdown or silencing of the expression of those genes (1).

We are currently using gene-specific double-stranded RNAs that are composed of two 21-nt long RNA strands that anneal in such a way as to leave 2-nt single-stranded overhangs at the 3'-end of each strand.  These are introduced into cultured cells using available transfection agents.  Suppression of gene expression can be demonstrated by Northern blot, RT-PCR or Western blot analysis.  We have successfully used RNAi to suppress expression of a transfected plasmid bearing a GFP-huntingtin fusion construct.

The facility can provide assistance with RNAi design and cell transfection for $100 per contact hour.

References:
(1) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.
Donze O. and Picard D. Nucleic Acids Res 2002 May 15:30(10):e46
 

IN SITU HYBRIDIZATION is an imaging method to visualize mRNA expression in tissues and cells.  A fluorophore-labeled mRNA probe that has consensus sequence to mRNA you are interested in, will be hybridized to the fixed tissues or cells.  The resultant fluorescence will be detected an imaged with a microscope.  Overall procedures include:
1. Synthesis (expression) of mRNA probe
2. Fluorescence0labeling of the mRNA probe
3. Fixation and hybridization of the specimen
4. Mounting of the specimen and microscopic imaging.

Charges:

Procedures 2-4 (10 slides) for OMRF investigators - $300
Procedures 2-4 (10 slides) for OUHSC investigators - $600
(These charges do not include the cost of synthesis of RNA probe)

Please contact Dr. Charles Stewart (Charles-Stewart@omrf.ouhsc.edu, 271-7583) for details.