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.

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 µl RNasin®

1 µl GACU pool

2 µl DTT

4 µl 5X transcription buffer

1 µl RPA Template Set

10 µl [a-32P]UTP

1 µl T7 RNA polymerase (Keep at -20℃ until use, return to -20℃ immediately).

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

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

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

26 µl 20 mM EDTA

25 µl Tris-saturated phenol

25 µl chloroform:isoamyl alcohol (50:1)

2 µl 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 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 tube and add 50 µl 4 M ammonium acetate and 250 µl ice cold 100% ethanol. Invert the tube to mix and incubate for 30 minutes at -70℃. Spin in a microfuge for 15 minutes at 4℃.

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℃.

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 µl of hybridization buffer and solubilize the pellet by gently vortexing for 20 seconds and quick spin on microfuge.

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