RNase Protection Assay

RNase Protection Assay发布日期:2006-8-3 热门指数:3608 收藏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 orradiolabeled 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 theappropriately-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, withoutfurther purification of poly-A+ RNA.PharMingen's RiboQuant TM Multi-Probe RNase Protection Assay is a complete system available for detecting and quantifying transcripts. This system includes:Multi-Probe Template SetIn Vitro Transcription KitRPA KitIndividual 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 VitroTranscription Kit, and the RPA Kit.Standard RPA ProcedureIn all steps of the protocol, standard precautions should be used to avoidRNase contamination and exposure of personnel to radioactivity. Typically,the probe synthesis is performed during the afternoon Day 1, hybridizationsare incubated overnight, and RNase treatments and gel electrophoresis areperformed early on Day 2.Probe Synthesis:1.Bring the [a-32P]UTP, GACU nucleotide pool, DTT, 5X transcriptionbuffer, and RPA template set to RT. For each probe synthesis, addthe following in order to a 1.5 ml Eppendorf tube:1 µl RNasin®1 µl GACU pool2 µl DTT4 µl 5X transcription buffer1 µl RPA Template Set10 µl [a-32P]UTP1 µl T7 RNA polymerase (Keep at -20°C until use, return to -20°Cimmediately).Mix by gentle pipetting or flicking and quick spin in a microfuge.Incubate at 37°C for 1 hour.2.Terminate the reaction by adding 2 µl of DNase. Mix by gentle flickingand quick spin in a microfuge. Incubate at 37°C for 30 minutes.3.Add the following reagents (in order) to each 1.5 ml Eppendorf tube:26 µl 20 mM EDTA25 µl Tris-saturated phenol25 µl chloroform:isoamyl alcohol (50:1)2 µl yeast tRNAMix by vortexing into an emulsion and spin in a microfuge for 5minutes at RT.4.Transfer the upper aqueous phase to a new 1.5 ml Eppendorf tubeand add 50 µl 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 tubeand add 50 µl 4 M ammonium acetate and 250 µl ice cold 100%ethanol. Invert the tube(s) to mix and incubate for 30 minutes at -70°C. Spin in a microfuge for 15 minutes at 4°C.6.Carefully remove the supernatant and add 100 µl of ice cold 90%ethanol to the pellet. Spin in a microfuge for 5 minutes at 4°C.7.Carefully remove the supernatant and air dry the pellets for 5 to 10minutes (do not dry in a vacuum evaporator centrifuge). Add 50 µl of hybridization buffer and solubilize the pellet by gently vortexing for 20seconds and quick spin on microfuge.8.Quantitate duplicate 1 µl samples in the scintillation counter. Expecta maximum yield of 1-3 x 106 Cherenkov counts / µl (measurement ofcpm / µl without the presence of scintillation fluid) with an acceptable lower limit of 3 x 105 Cherenkov counts / µl. Store the probe at -20°C until needed. Generally, the probe can be used for two successiveovernight hybridizations at most.RNA Preparation & Hybridization:9.For the best results, use procedures that generate total RNA of highquality and purity. RNA should be stored in RNase-free water at-70°C. Add the desired amount of target RNA (generally 1-20 µg) to1.5 ml Eppendorf tubes and include a tube that contains yeast tRNAas 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 µl volume (i.e., 2 µg) is recommended.10.If RNA has been stored in water, freeze the samples for 15 minutesat -70°C. Dry completely (~1 hour) in a vacuum evaporator centrifuge (no heat). Likewise, RNA can be precipitated prior to the addition ofhybridization buffer as in Step 5.11.Add 8 µl of hybridization buffer to each sample. Solubilize the RNAby gently vortexing for 3-4 minutes and quick spin in the microfuge.12.Dilute the probe from Step 8 with hybridization buffer to theappropriate concentration: Add 2 µl of diluted probe to each RNAsample and mix by pipetting. Add a drop of mineral oil to each tubeand quick spin in the microfuge.13.Place the samples in a heat block pre-warmed to 90°C. Immediatelyturn the temperature to 56°C (allowing the temperature to ramp down slowly) and incubate for 12-16 hours. Turn the heat block to 37°C for15 minutes prior to the RNase treatments, again allowing thetemperature to ramp down slowly. All incubations may also becarried out in a water bath.RNase Treatments:14.Prepare the RNase cocktail (per 20 samples)2.5 ml RNase buffer6 µl RNase A + T1 mixRemove the RNA samples from the heat block and pipet 100 µl of the RNase cocktail underneath the oil into the aqueous layer (bubble).Spin in microfuge for 10 seconds and incubate for 45 minutes at30°C.15.Before the RNase digestion is completed, prepare the Proteinase Kcocktail (per 20 samples):390 µl Proteinase K buffer30 µl Proteinase K30 µl yeast tRNAMix and add 18 µl aliquots of the cocktail to new Eppendorf tubes.ing 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, andincubate for 15 minutes at 37°C.17.Add 65 µl Tris-saturated phenol and 65 µl chloroform:isoamyl alcohol(50:1). Vortex into an emulsion and spin in the microfuge for 5minutes at RT.18.Carefully extract the upper aqueous phase (set the pipettor at 120 µland totally avoid the organic interface) and transfer to a new tube.Add 120 µl 4 M ammonium acetate and 650 µl ice cold 100%ethanol. Mix by inverting the tubes and incubate for 30 minutes at -70°C. Spin in the microfuge for 5 minutes at 4°C.19.Carefully remove the supernatant and add 100 µl ice cold 90%ethanol. Spin in the microfuge for 5 minutes at 4°C.20.Carefully remove the supernatant and air dry the pellet (do not dry ina vacuum evaporator centrifuge). Add 5 µl of 1X loading buffer,vortex for 2-3 minutes, and quick spin in the microfuge. Prior toloading the samples on the gel, heat the samples for 3 minutes at 90°C 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 waterfollowed by ethanol. Siliconize the short plate and clean again.Assemble the gel mold (0.4 mm spacers).bine 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% acrylamide9.31 mls of 2% bis acrylamide7.45 mls of 10x TBE35.82 g of UreaQS to 74.5 ml with dH2O450 µl ammonium persulfate (10%)60 µl TEMEDPour immediately into the gel mold, remove any air bubbles, and add an appropriate comb (e.g., 5 mm well width). Use of a sharks toothcomb is not recommended.23.After polymerization (~1 hour), remove the comb and flush the wellsthoroughly with 0.5X TBE. Place each gel in a vertical rig (use a gelset up that has a heat dispenser) and prerun at 40 watts constantpower for ~ 45 minutes, with 0.5X TBE as the running buffer. Geltemperature should be 50°C.24.Flush the wells again with 0.5X TBE and load the samples (from Step20). Also load a dilution of the probe set in loading buffer (typically1000-2000 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 thegel 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 80°C. Place the dried gel on film (Kodak X-AR) in a cassette with an intensifying screen and develop at -70°C (Exposure times will vary depending on application). Alternatively, radioactivity can be quantified by phosphorimaging or other equivalentinstruments.26.With the undigested probes as markers, plot a standard curve on asemi-log graph paper, of migration distance versus log nucleotidelength. Use this curve to establish the identity of "RNase-protected"bands in the experimental samples. Note that the probe lengths aregreater than the "protected" fragment lengths, this is due to thepresence of flanking sequences in the probes that are derived from the plasmid and do not hybridize with target mRNA.Technical Tips:Poor probe recoveries.e of [a-32P]UTP that has decayed beyond one half life may lead todecreased probe labeling and increased lane background. Werecommend the use of [a-32P]UTP which does not containcommercial stabilizers.B.Avoid repeated freeze-thaw of the DTT stock solution. Werecommend storing small aliquots at -20°C.C.Make sure that the transcription reagents (nucleotides, DTT, and 5Xtranscription buffer) are at RT prior to adding RPA template.Spermidine present in the transcription buffer can precipitate DNA atlow temperatures.D.Careless removal of ethanol from the precipitated probe can lead tosignificant losses (we have included yeast tRNA as a carrier to facilitate precipitation). If this problem is suspected, recentrifuge theethanol supernatant.E.Check the integrity of the probe set by analyzing it on acrylamide gel.Note: High level of certain mRNA species obscure detection ofother, rarer mRNA species.F.Consult Pharmingen for the availability of RPA template setscustomized to omit probes for the highly expresses transcripts in yourRNA preparations.High levels of breakdown products in the gel lanes.A. A reasonable level of protected probe fragmentation is normalbecause 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 RPAtemplate sets.e caution when extracting the aqueous phase from thephenol-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-onlyextraction.。