Non-radioactive ribonuclease Protection Assay

The ribonuclease protection assay (RPA) is a highly sensitive method for quantification of mRNA. The synthesis and labeling of several apoptotic genes is done using templates from the RiboQuant system (PharMingen). This method has the advantage that allows the comparative analysis of different mRNA species within the samples and, by incorporating probes for housekeeping transcripts, we can compare the level of each individual mRNA species. We have characterized a non-radioactive form of the conventional RPA based on using biotinylated nucleotides and a highly sensitive chemiluminiscent detection system.

Reagents
1.10X Biotin RNA labeling Mix- Boehringer, Cat# 1685 597
2.Hy-bond (+) charged nylon membranes- Amersham Cat # RPN303B
3.Avidin-Alkalin Phosphatase-Tropix, Cat # MS050R
4.Chemiluminescent Hyperfilm-Amersham Cat # RPN2103
5.Tris-saturated phenol.
6.Chloroform:isoamyl alcohol (50:1)
7.95% Ethanol
8.40% acrylamide/5% Bis solution
9.Urea
10.DEPC water
Buffers and Media
1.10X TBE in 500 ml DEPC water: 0.89 M Tris (54 g); 0.89M Boric Acid (28 g); 20 mM EDTA (3g)
2.Washing Buffer (prepare stock 2X): 400 ml 10X PBS; 200 ml 10X SDS; up to 2 liters with water. The day of use prepare 1X solution.
3.10X Assay Buffer: 200 mM Tris HCl (pH 9.8); 10 mM Magnesium Chloride
Store at 4°C. Prepare every time a fresh 1X solution. You may need 100 ml.
Blocking Buffer
Final Concentration
Stock
0.2% Blocking Reagent0.4 g
1X PBS20 ml (10x PBS)
0.5 % SDS10 ml (10% SDS
DDWUp to 200 ml
Cell lines
1.Jurkat cell line: T cells (Target)
2.JAR cell line: Choriocarcinoma cells (Effector)
Probe Synthesis
1.Pre-warm heat block to 37°C
2.Bring Biotin 10X labeling mix, DTT, 5X Transcription buffer, and RPA template set to room temperature. Add the following to 2 µl 10X labeling mix: 2 µl DTT, 1 µl RPA Template set, 4 µl, 5X transcription buffer, 1 µl RNAsin, 1 µl T7 polymerase, 9 ml DEPC H2O. Total volume: 20 µl
3.Mix by gentle pipetting and quick spin in microfuge. Incubate at 37°C for 1 hour. Thaw DNase at this point.
4.Terminate reaction by adding 2 µl DNase. Mix by gentle flicking and quick spin in microfuge. Incubate 37oC for 30 min.
5.Add in order: 26 µl 20mM EDTA, 50 µl Tris-saturated phenol/Chloroform: isoamyl alcohol, 2 µl yeast tRNA.
6.Mix by vortex into an emulsion and spin 12,000 g X 5-min room temp.
7.Transfer upper aqueous phases to a new Eppendorf tube and add 50 µl Chloroform: isoamyl alcohol.
8.Mix by vortex and spin in microcentrifuge (12,000g) for 2-min room temp.
9.Transfer the upper aqueous phase to a new Eppendorf tube and add 50 µl 4M ammonium acetate and 250 µl ice cold 95% ethanol. Invert tubes to mix and incubate for 30 min at -70°C. Spin in microfuge (12,000x g) for 5 min at 4°C.
10.Carefully remove the supernatant and add 100 µl ice cold 95% ethanol to the pellet. Spin in a microfuge (12,000x g) for 5 min at 4oC.
11.Carefully remove the entire supernatant and air dry pellet for 5-10 min. Resuspend the pellet in 50 µl hybridization buffer by gently vortexing for 20 seconds. Quick spin in microfuge. Store probes as 5 µl aliquots at -70°C until needed.
RNA Hybridization
1.Add 10 µl total RNA to separate Eppendorf tubes. Freeze sample for 15 min at -70°C. Dry completely (~40 min) in a vacuum evaporator centrifuge (no heat).
2.Add 8 µl hybridization buffer to each sample. Solubilize RNA by gently vortexing for 3-4 min and quick spin in microfuge.
3.Add 2 µl of probe set to each RNA sample, and mix by pipetting. Add two drops of mineral oil to each tube and quick spin in microfuge.
4.Place samples in a heat block pre-warmed to 90°C. Immediately turn the temperature to 56°C and incubate for 12-16 h. Turn the heat block to 37°C for 15 min prior to the RNase treatment.
RNase Treatment
1.Prepare the RNase cocktail (per 10 samples): 1,25 ml RNase buffer, 3 µl RNase A + T1 mix
2.Remove the RNA samples from the heat block and add 100 µl of the RNase cocktail underneath the oil. Spin in microfuge for 15 sec and incubate for 45 min at 30°C. When done set heat block to 37°C.
3.Before the RNase digestion is completed, prepare the proteinase K cocktail (per 10 samples): 195 µl proteinase K buffer, 15 µl proteinase K, 15 µl yeast tRNA.
4.Mix and add 18 ml aliquots to fresh Eppendorf tubes.
RNA Purification
1.Set pipette to 105 µl, extract the RNase digests from underneath the oil (avoid the oil) and transfer to the tubes containing the proteinase K solution. Quick vortex and spin in microfuge and incubate for 15 min at 37°C.
2.Add 130 µl Tris-saturated phenol/Chloroform: isoamyl alcohol. Vortex into an emulsion and spin in microfuge (12,000x g) for 5 min at room temperature.
3.Carefully extract the upper aqueous phase (Set pipette at 120 µl and totally avoid the organic interface) and transfer to a new tube. Add 120 µl 4M ammonium acetate and 650 µl ice cold 95% ethanol. Mix by inversion and incubate 30 min at -70°C.
4.Centrifuge the samples at 12,000x g for 15 min at 4°C.
5.Carefully remove the supernatant and add 100 µl ice cold 95% ethanol. Spin in microfuge (12,000x g) for 5 min at 4°C.
6.Carefully remove the supernatant and air-dry the pellet completely. Add 2 µl 1X loading buffer, vortex 2-3 min, and quick spin in the microfuge. Prior to loading samples, heat them for 3 min at 90°C and place immediately on an ice bath.
Gel Resolution of Protected Probes
1.Make two dilution of the 50X QP running buffer: Solution A: 65 ml of 5X QP running buffer (1:10 dilution); Solution B. 750 ml of 1X QP running buffer (1:50 dilution)
2.Heat 750 ml 1X running buffer to 50-60°C Assemble QuickPoint Cell: a). Thoroughly wash wells of gel with water; b). Place gels in chamber with shorter glass towards buffer core (if only running one gel use buffer dam in place of second gel); c. Place upper wedge, but do not press down yet.
3.Fill outer chamber with heated Solution B. Press down firmly on rear wedge to set it and seal chamber.
4.Fill inner chamber with unheated Solution A
5.Pre-run the gel at 1200 volts for 5 min.
6.Load samples, 1.5 µl/well.
7.Run gel at 1200 volts until upper loading dye runs _ of the gel (~10 min)
Blotting of Gel

1.

Cut a positively charged nylon membrane (Hyband N+) to 11cm X 8cm and mark the top side to indicate the side with transferred RNA. Wet the membrane with water and drain excess water off.

2.

Use razor blade to cut open tape on side of gel. Pry glass plates apart and immediately lay membrane on top of gel. Use glass pipette to remove all bubbles. Place two pieces of dry filter paper on top of the membrane. Place the remaining glass plate on top of filter paper. Place a 1 liter bottle filled with water on top of the glass plate and allow capillary transfer of the RNA to proceed for 20 min.

3.

Remove the membrane from the transfer; rinse with water to remove gel pieces.

4.

Dry the membrane 5-10 min at RT.

5.

Fix the membrane by UV crosslinking (1200 joules for 2 min).