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P r o t o c o l s

RNase Protection Assay

 

The ribonuclease protection assay (RPA) is a highly sensitive and specific method for the detection of mRNA species. The assay was made possible by the discovery and characterization of DNA-dependant RNA polymerases from the bacteriophages SP6, T7 and T3, and the elucidation of their cognate promoter sequences. These polymerases are ideal for the synthesis of high-specific-activity RNA probes from DNA templates because these polymerases exhibit a high degree of fidelity for their promoters, polymerize RNA at a very high rate, efficiently transcribe long segments, and do not require high concentrations of rNTPs. Thus a cDNA fragment of interest can be subcloned into a plasmid that contains bacteriophage promoters, and the construct can then be used as a template for synthesis or radiolabeled anti-sense RNA probes.

 

Pharmingen's strategy for the development of multi-probe RPA systems is to generate a series of apoptosis-related gene templates, each of distinct length and each representing a sequence in a distinct mRNA species. The templates are assembled into biologically relevant sets to be used by investigators for the T7 polymerase-directed synthesis of a high-specific-activity, [32P]-labeled antisense RNA probe set. The probe set is hybridized in excess to target RNA in solution after which free probe and other single-stranded RNA are digested with RNases. The remaining "RNase-protected" probes are purified, resolved on denaturing polyacrylamide gel, and quantified by autoradiography or phosphorimaging. The quantity of each mRNA species in the original RNA sample can then be determined based on the intensity of the appropriately-sized, protected probe fragment.

 

Two distinct advantages of the multi-probe RPA approach are its sensitivity and its capacity to simultaneously quantify several mRNA species, in a single sample of total RNA. This allows comparative analysis of different mRNA species within samples and, by incorporating probes for housekeeping gene transcripts, the levels of individual mRNA species can be compared between samples. Moreover,the assay is highly specific and quantitative due to the RNase sensitivity of mismatched base pairs and the use of solution-phase hybridization driven toward completion by excess probe. Lastly, the multi-probe RPA can be preformed on total RNA preparations derived by standard methods from either frozen tissues or cultured cells, without further purification of poly-A+ RNA.

 

PharMingen's RiboQuantTM Multi-Probe RNase Protection Assay is a complete system available for detecting and quantifying transcripts. This system includes:


Multi-Probe Template Set
In Vitro Transcription Kit
RPA Kit

 

Individual components may be purchased either separately or together as the RPA Starter Package (Cat. No. 45024K). The starter package includes one Template Set, the In Vitro Transcription Kit, and the RPA Kit.

 

Standard RPA Procedure

 


In all steps of the protocol, standard precautions should be used to avoid RNase contamination and exposure of personnel to radioactivity. Typically, the probe synthesis is performed during the afternoon Day 1, hybridizations are incubated overnight, and RNase treatments and gel electrophoresis are performed early on Day 2.

Probe Synthesis:

 

1.

 

Bring the [a-32P]UTP, GACU nucleotide pool, DTT, 5X transcription buffer, and RPA template set to RT. For each probe synthesis, add the following in order to a 1.5 ml Eppendorf tube:

1 l RNasin
1 l GACU pool
2 l DTT
4 l 5X transcription buffer
1 l RPA Template Set
10 l [a-32P]UTP
1 l T7 RNA polymerase (Keep at -20C until use, return to -20C immediately).

Mix by gentle pipetting or flicking and quick spin in a microfuge. Incubate at 37C for 1 hour.

 

 

 

 

 

2.

 

Terminate the reaction by adding 2 l of DNase. Mix by gentle flicking and quick spin in a microfuge. Incubate at 37C for 30 minutes.

 

 

 

 

 

3.

 

Add the following reagents (in order) to each 1.5 ml Eppendorf tube:

26 l 20 mM EDTA
25 l Tris-saturated phenol
25 l chloroform:isoamyl alcohol (50:1)
2 l yeast tRNA

Mix by vortexing into an emulsion and spin in a microfuge for 5 minutes at RT.

 

 

 

 

 

4.

 

Transfer the upper aqueous phase to a new 1.5 ml Eppendorf tube and add 50 l chloroform:isoamyl alcohol (50:1). Mix by vortexing, then spin in a microfuge for 2 minutes at RT.

 

 

 

 

 

5.

 

Transfer the upper aqueous phase to a new 1.5 ml Eppendorf tube and add 50 l 4 M ammonium acetate and 250 l ice cold 100% ethanol. Invert the tube(s) to mix and incubate for 30 minutes at -70C. Spin in a microfuge for 15 minutes at 4C.

 

 

 

 

 

6.

 

Carefully remove the supernatant and add 100 l of ice cold 90% ethanol to the pellet. Spin in a microfuge for 5 minutes at 4C.

 

 

 

 

 

7.

 

Carefully remove the supernatant and air dry the pellets for 5 to 10 minutes (do not dry in a vacuum evaporator centrifuge). Add 50 l of hybridization buffer and solubilize the pellet by gently vortexing for 20 seconds and quick spin on microfuge.

 

 

 

 

 

8.

 

Quantitate duplicate 1 l samples in the scintillation counter. Expect a maximum yield of 1-3 x 106 Cherenkov counts / l (measurement of cpm / l without the presence of scintillation fluid) with an acceptable lower limit of 3 x 105 Cherenkov counts / l. Store the probe at -20C until needed. Generally, the probe can be used for two successive overnight hybridizations at most.

RNA Preparation & Hybridization:

 

9.

 

For the best results, use procedures that generate total RNA of high quality and purity. RNA should be stored in RNase-free water at -70C. Add the desired amount of target RNA (generally 1-20 g) to 1.5 ml Eppendorf tubes and include a tube that contains yeast tRNA as a background control. In general, 20-24 total sample tubes are an easily manageable number for each RPA setup. With the Pharmingen control RNA, 2 l volume (i.e., 2 g) is recommended.

 

 

 

 

 

10.

 

If RNA has been stored in water, freeze the samples for 15 minutes at -70C. Dry completely (~1 hour) in a vacuum evaporator centrifuge (no heat). Likewise, RNA can be precipitated prior to the addition of hybridization buffer as in Step 5.

 

 

 

 

 

11.

 

Add 8 l of hybridization buffer to each sample. Solubilize the RNA by gently vortexing for 3-4 minutes and quick spin in the microfuge.

 

 

 

 

 

12.

 

Dilute the probe from Step 8 with hybridization buffer to the appropriate concentration: Add 2 l of diluted probe to each RNA sample and mix by pipetting. Add a drop of mineral oil to each tube and quick spin in the microfuge.

 

 

 

 

 

13.

 

Place the samples in a heat block pre-warmed to 90C. Immediately turn the temperature to 56C (allowing the temperature to ramp down slowly) and incubate for 12-16 hours. Turn the heat block to 37C for 15 minutes prior to the RNase treatments, again allowing the temperature to ramp down slowly. All incubations may also be carried out in a water bath.

RNase Treatments:

 

14.

 

Prepare the RNase cocktail (per 20 samples)

2.5 ml RNase buffer
6 l RNase A + T1 mix

Remove the RNA samples from the heat block and pipet 100 l of the RNase cocktail underneath the oil into the aqueous layer (bubble). Spin in microfuge for 10 seconds and incubate for 45 minutes at 30C.

 

 

 

 

 

15.

 

Before the RNase digestion is completed, prepare the Proteinase K cocktail (per 20 samples):

390 l Proteinase K buffer
30 l Proteinase K
30 l yeast tRNA

Mix and add 18 l aliquots of the cocktail to new Eppendorf tubes.

 

 

 

 

 

16.

 

Using a pipettor, extract the RNase digests from underneath the oil (try to avoid the oil) and transfer to the tubes containing the Proteinase K solution. Quick vortex, quick spin in the microfuge, and incubate for 15 minutes at 37C.

 

 

 

 

 

17.

 

Add 65 l Tris-saturated phenol and 65 l chloroform:isoamyl alcohol (50:1). Vortex into an emulsion and spin in the microfuge for 5 minutes at RT.

 

 

 

 

 

18.

 

Carefully extract the upper aqueous phase (set the pipettor at 120 l and totally avoid the organic interface) and transfer to a new tube. Add 120 l 4 M ammonium acetate and 650 l ice cold 100% ethanol. Mix by inverting the tubes and incubate for 30 minutes at -70C. Spin in the microfuge for 5 minutes at 4C.

 

 

 

 

 

19.

 

Carefully remove the supernatant and add 100 l ice cold 90% ethanol. Spin in the microfuge for 5 minutes at 4C.

 

 

 

 

 

20.

 

Carefully remove the supernatant and air dry the pellet (do not dry in a vacuum evaporator centrifuge). Add 5 l of 1X loading buffer, vortex for 2-3 minutes, and quick spin in the microfuge. Prior to loading the samples on the gel, heat the samples for 3 minutes at 90C and then place them immediately in an ice bath.

Gel Resolution of Protected Probes:

 

21.

 

Clean a set of gel plates (> 40 cm in length) thoroughly with water followed by ethanol. Siliconize the short plate and clean again. Assemble the gel mold (0.4 mm spacers).

 

 

 

 

 

22.

 

Combine the following to give a final concentration of 5% acrylamide:
74.5 ml acrylamide solution (final 19:1 acrylamide/bis):

8.85 mls of 40% acrylamide
9.31 mls of 2% bis acrylamide
7.45 mls of 10x TBE
35.82 g of Urea
QS to 74.5 ml with dH2O
450 l ammonium persulfate (10%)
60 l TEMED

Pour immediately into the gel mold, remove any air bubbles, and add an appropriate comb (e.g., 5 mm well width). Use of a sharks tooth comb is not recommended.

 

 

 

 

 

23.

 

After polymerization (~1 hour), remove the comb and flush the wells thoroughly with 0.5X TBE. Place each gel in a vertical rig (use a gel set up that has a heat dispenser) and prerun at 40 watts constant power for ~ 45 minutes, with 0.5X TBE as the running buffer. Gel temperature should be 50C.

 

 

 

 

 

24.

 

Flush the wells again with 0.5X TBE and load the samples (from Step 20). Also load a dilution of the probe set in loading buffer (typically 1000-2000 cpm/lane) to server as size markers. Run the gel at 50 watts constant power until the leading edge of the Bromophenol Blue (BPB) (front dye) reaches 30 cm.

 

 

 

 

 

25.

 

Disassemble the gel mold, remove the short plate, and absorb the gel to filter paper. Cover the gel with Saran wrap and layer between two additional pieces of filter paper. Place in the gel dryer vacuum for ~ 1 hour at 80C. Place the dried gel on film (Kodak X-AR) in a cassette with an intensifying screen and develop at -70C (Exposure times will vary depending on application). Alternatively, radioactivity can be quantified by phosphorimaging or other equivalent instruments.

 

 

 

 

 

26.

 

With the undigested probes as markers, plot a standard curve on a semi-log graph paper, of migration distance versus log nucleotide length. Use this curve to establish the identity of "RNase-protected" bands in the experimental samples. Note that the probe lengths are greater than the "protected" fragment lengths, this is due to the presence of flanking sequences in the probes that are derived from the plasmid and do not hybridize with target mRNA.

Technical Tips:

Poor probe recoveries.

 

A.

 

Use of [a-32P]UTP that has decayed beyond one half life may lead to decreased probe labeling and increased lane background. We recommend the use of [a-32P]UTP which does not contain commercial stabilizers.

 

 

 

 

 

B.

 

Avoid repeated freeze-thaw of the DTT stock solution. We recommend storing small aliquots at -20C.

 

 

 

 

 

C.

 

Make sure that the transcription reagents (nucleotides, DTT, and 5X transcription buffer) are at RT prior to adding RPA template. Spermidine present in the transcription buffer can precipitate DNA at low temperatures.

 

 

 

 

 

D.

 

Careless removal of ethanol from the precipitated probe can lead to significant losses (we have included yeast tRNA as a carrier to facilitate precipitation). If this problem is suspected, recentrifuge the ethanol supernatant.

 

 

 

 

 

E.

 

Check the integrity of the probe set by analyzing it on acrylamide gel.
Note: High level of certain mRNA species obscure detection of other, rarer mRNA species.

 

 

 

 

 

F.

 

Consult Pharmingen for the availability of RPA template sets customized to omit probes for the highly expresses transcripts in your RNA preparations.

High levels of breakdown products in the gel lanes.

 

A.

 

A reasonable level of protected probe fragmentation is normal because mRNA degradation is a natural occurrence within cells. However, if excessive degradation is observed, check the integrity of your RNA samples by gel electrophoresis.

 

 

 

 

 

B.

 

Rigorously adhere to the prescribed RNase digestion conditions. These have been carefully optimized for the Pharmingen RPA template sets.

 

 

 

 

 

C.

 

Use caution when extracting the aqueous phase from the phenol-chloroform extraction (Step 18) because residual RNase may be present in the organic interface. This problem can be remedied by performing a second phenol-chloroform or chloroform-only extraction.

 

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