Protocols

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°C until use, return to -20°C immediately).

2.

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

3.

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

4.

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

5.

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

6.

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.

7.

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(s) to mix and incubate for 30 minutes at -70°C. Spin in a microfuge for 15 minutes at 4°C.

8.

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.

9.

Carefully remove all 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 30 seconds. Quick spin in a microfuge.

10.

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 -80°C until needed. Generally, the probe can be used for two successive overnight hybridizations at most.

1.

For the best results, use procedures that generate total RNA of high quality and purity. RNA should be stored in RNase-free water at -70°C. Add the desired amount of target RNA (generally 1-20 µg) to 1.5 ml Eppendorf tubes and include a tube that contains yeast tRNA as a background control. Include 2 µl of the appropriate BD RiboQuant Control RNA as a positive control, and a tube containing 2 µl of 2 mg/ml tRNA as a background control. In general, 20-24 total sample tubes are an easily manageable number for each RPA setup.

2.

If RNA has been stored in water, freeze the samples for 15 minutes at -70°C. 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 7.

3.

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

4.

Dilute the probe from Step 10 with hybridization buffer to the appropriate concentration. The optimal probe concentration (cpm/µl) for each standard Multi-Probe Template Set is included on the technical data sheet supplied with the set. Add 2 µl 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.

5.

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

1.

.

Prepare the RNase cocktail (per 20 samples)

2.5 ml RNase buffer
6 µl RNase A + T1 mix

2.

Remove 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 at 30°C.

3.

During the RNase digestion, prepare the Proteinase K cocktail. Prewarm Proteinase K buffer to 37°C to solubilize SDS prior to using.

390 µl Proteinase K buffer
30 µl Proteinase K
30 µl yeast tRNA

Mix and add 18 µl aliquots of the cocktail to new, labeled Eppendorf tubes.

4.

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. Vortex briefly, quick spin in the microfuge, and incubate for 15 minutes at 37°C.

5.

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

6.

Carefully extract the upper aqueous phase (set the pipettor at 120 µl and 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 20 minutes at 4°C.

7.

Carefully remove the supernatant and add 100 µl ice cold 90% ethanol. Spin in the microfuge for 10-15 minutes at 4°C.

8.

Carefully remove the supernatant and air dry the pellet (do not dry in a vacuum evaporator centrifuge). Add 5 µl 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 90°C and then place them immediately in an ice bath.

1.

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).

2.

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 µl ammonium persulfate (10%)
60 µl TEMED

Note: Do not use commercially available, pre-mixed acrylamide/bis solutions or pre-cast gels. Use recommended acrylamide concentration and acrylamide:bis ratio. It is critical for the correct resolution of unprotected and protected probe bands.

3.

Mix acrylamide solution well, pour immediately into the gel mold, remove any air bubbles, and insert an appropriate comb (e.g., 5 mm well width). Do not use a sharks tooth comb.

4.

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 50°C.

5.

Flush the wells with running buffer (0.5X TBE). Load the samples and controls, including ³²P-labeled probe, diluted to 1000-2000 cpm in 10 µl loading buffer to serve as size markers. Run the gel at 55 watts constant power until the leading edge of the Bromophenol Blue (BPB) (front dye) reaches 30 cm from the bottom of the well.

6.

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 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 equivalent instruments.

7.

Using 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.

1.

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

2.

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

3.

Make certain 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.

4.

To maximize recovery of probe, orient hinge of microfuge tube in direction of centrifugal force in microfuge, and remove tube from microfuge immediately following spin. Carefully remove ethanol and ethanol washes without touching or dislodging pellet. Following ethanol wash, briefly centrifuge for 10 sec. and remove residual ethanol with a P200 pipet tip.

5.

Careful removal of ethanol from the precipitated probe will reduce probe loss (we have included yeast tRNA as a carrier to facilitate precipitation). If this problem is suspected, refreeze and recentrifuge the ethanol supernatant.

6.

Check the integrity of the probe set by analyzing it on an acrylamide gel.

7.

Quantitate duplicate 1 µl samples in the scintillation counter. Expect a maximum yield of ~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°C until needed. Generally, the probe can only be used for two successive overnight hybridizations (when labeled with [a-32P] UTP). Probe pellet can be resuspended in 25 µl (vs. 50 µl) to obtain a greater cpm/µl concentration.

1.

Use recommended polyacrylamide concentration and bis/acrylamide ratio.

2.

Immediately prior to loading, denature sample for a full three min at 90°C in a heat block (not oven), then place samples immediately on ice.

1.

Some 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.

2.

Rigorously adhere to the prescribed RNase digestion conditions. These have been carefully optimized for the BD RiboQuant RPA Multi-Probe Template Sets.

3.

Use caution when extracting the aqueous phase from the phenol-chloroform extraction (Step 6 of RNAse treatment) 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.

4.

Residual phenol or ethanol in the sample will cause the appearance of degradation of the probe and is characterized by narrowing of the lanes in the lower portion of the gel.

5.

Radiolysis of labeled probe stored over time will contribute to high background.