Endy:RNase Protection Assay

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Samples to be probed

1. Experimental Samples
   • Total Cellular RNA ~10 ug total cellular RNA, would contain 10 fg - 1 pg of rare RNA, ~300 pg of moderately abundant RNA.
2. In Vitro Standard Curve Samples
   • RNA In Vitro Standards 1 fg - 100 pg range suggested. (Molecular Cloning) 1 - 50 ng for high copy/plasmid-based, In carrier RNA to constant total mass, ~10 ug.
3. Molecular Weight Standard
   • 32P End-Labeled DNA Ladder

Precipitation and Hybridization

1. Combine:
   • Sample or IVT standard RNA
   • Carrier RNA to equivalent total mass RNA. (Stock solutionn yeast tRNA = 1ug/uL)
   • H2O to 99 uL total volume
   • 10 uL (0.1volume) of 3M sodium acetate (pH 5.2)
   • 1 uL Riboprobe (suggested 2x105 to 10x105 cpm, 0.1-0.5 ng) NOTE: may need significantly more to be in excess (cmc, 4-5-05)
   • 250 uL (2.5 volume) ice-cold 100% EtOH
2. Incubate 10 min at -80° C (Optional, 20 minLonger incubation/spin yields better recovery of short or low quantity RNA.)
3. Centrifuge 10 min at 4° C (Optional, 25 min. Longer incubation/spin yields better recovery of short or low quantity RNA.)
4. Decant and wash pellet with 1mL of cold 80% EtOH
5. Decant. Incubate at room temp until all EtOH has evaporated, ~15 min.
6. Dissolve Samples in 30 uL hybridizaton buffer. Pipette numerous times to dissolve pellet. Roll bead of buffer across tube wall where RNA is deposited. (Heat to 60° C if difficult to resuspend RNA.)
7. Transfer samples to pcr tubes
8. Run Hybridization Program on PCR block:
   • 10 min at 85° C to denature RNAs
   • 8-12 hours at annealing temp (specific to probe, usually 45-50° C, determine optimal temp experimentally by hybridization with IVT at 25-65° C.)
   • Cool to 22.5° C (optional, cool to 4° C for 10-20 min. May stabilize hybrids prior to digest.)

RNase Digest

1. Prepare RNase digestion mixture. RNases must be added fresh just before digest.
   1. For 1mL RNase digestion mixture, combine:
      • 994 uL 1x buffer (300mM NaCl, 10mM Tris-Cl, pH 7.4, 5mM EDTA, pH 7.5)
      • 4 uL of RNase A (10 ug/uL in 10mM Tris-Cl, 15mM NaCl)
      • 1 uL RNase T1 (100 units/uL in 10mM Tris-Cl, 15mM NaCl)
2. Transfer samples to Eppendorf tubes.
3. Add 300 uL RNase digestion mixture per sample. Incubate 60 min at 30° C. (Variable if signal to noise is poor after autoradiography.)
4. Add 20 uL of 10% SDS and 5 uL of 20 mg/mL proteinase K to stop the reaction. Incubate 30 min at 37° C.

EtOH Precipitation

1. Combine samples with
   • 32 uL (0.1volume) of 3M sodium acetate (pH 5.2)
   • 800 uL (2.5 volume) ice-cold 100% EtOH
2. Incubate 10 min at -80° C (Optional, 20 minLonger incubation/spin yields better recovery of short or low quantity RNA.)
3. Centrifuge 10 min at 4° C (Optional, 25 min. Longer incubation/spin yields better recovery of short or low quantity RNA.)
4. Decant and wash pellet with 1mL of cold 80% EtOH.
5. Decant. Incubate at room temp until all EtOH has evaporated, ~15 min.

Gel Analysis

1. Add 10 uL RNA gel-loading buffer (with formamide) to samples and MW marker. (Use ~0.1 - 1 uL MW marker, depending on activity.)
2. Incubate samples 5 min at 95° C. Transfer immediately to ice bath. Pulse-spin to consolidate sample.
3. Load samples and markers in 6% polyacrylamide/8M urea gel, 0.75 mm thickness in 1x TBE buffer. See note on standard curves.
4. Electrophoresis: Run at 280 V for 7 cm mini-gel. Note: Glass plates should be warm to touch, usually 40-45 V/cm.

dsRNA will run ~5-10% slower than DNA in this range; closer together at higher current.

1. Allow tracking dyes to run to appropriate distance.
   • Note: In a 6% polyacrylamide gel: Xylene Cyanol runs at 110 nt, Bromophenol Blue runs at 25 nt
2. Turn off power and dismantle electrophoresis set-up. Pry up one corner of the larger glass plate and cut one corner of the gel for orientation purposes.
3. Transfer glass plate with gel to flat bench top. Wisk off excess ddH20 by blotting edge with kimwipe. (Do not blot the gel surface.)
4. Transfer gel to Whatman 3 MM filter paper that is 1 cm larger than the gel on all sides by laying paper on top of gel and inverting all.
5. Remove glass plate, place saran wrap over gel, do not wrap underneath.
6. Dry the gel on a pre-heated gel dryer for 30 min at 80° C. Note: for higher percentage gels it is necessary to turn heat off prior to breaking the vacuum seal or gel will shatter.
7. While gel dries, erase Phosphorimager screen, 20 min on light table.
8. After removing gel from dryer, fold saran wrap around all.

Autoradiography

1. Place dry gel in Phorphorimager cassette, facing erased screen.
2. Incubate 1 hour
3. Image screen on phosphorimager (settings: 176 micron pixel value, pmt = 650)
4. Erase screen (20 min face down on Sauer Lab light table is sufficient.)
5. Repeat incubation overnight for higher signal intensity. Finish by erasing screen.

Image Analysis/ Quantitation

• Use ImageQuant

Additional Notes

1. Standard Curves: It's possible to condense samples from multiple standard curves in one lane, either by running samples of different sizes in the same lane, or by loading samples with different masses of standard in succession (if you do not care to see the MW of the standard.)

Buffers for Ribonuclease Protection Assay

Hybridization Buffer (w/ Formamide); 5 mL

from Molecular Cloning A1.12

• 200 uL 1M PIPES (40 mM)
• 10 uL 0.5M EDTA (1 mM)
• 400 uL 5M NaCl (0.4 M)
• 80% (v/v) deionized formamide: 4 mL

• pH to 6.4 w/ 1N HCl (~10 uL)
• adjust volume with nuclease-free water
• store 4° C wrapped in foil
• note: use disodium salt of PIPES (sesquisodium salt of PIPES worked fine also)

RNase Digestion Mix Buffer

• 300 mM NaCl
• 10 mM Tris-Cl (pH 7.4)
• 5 mM EDTA (pH 7.5)

• For 100 mL, combine:
  • 80 mL nuclease-free H2O
  • 10 mL of 3 M NaCl
  • 1 mL of 1 M Tris-Cl (pH 8.0)
  • 1 mL of 0.5 M EDTA
  • pH to 7.5 with 1N HCl, if necessary.
  • Adjust volume to 100 mL.

RNase Dilution Buffer

• 15 mM NaCl
• 10 mM Tris-Cl (pH 7.5)

• For 10 mL, combine:
  • 50 uL of 3 M NaCl
  • 100 uL of 1 M Tris-Cl (pH 8.0)
  • 9.85 mL nuclease-free H2O

RNA Gel Loading Buffer with Formamide; 10mL

NOTE: Store in the dark, at 4°C

• 9.5 mL of deionized formamide
• 100 uL of 0.5 mM EDTA, pH 8.0
• 2.5 ug of bromophenol blue
• 2.5 ug of xylene cyanol
• 2.5 ug SDS
• 400 uL nuclease-free H2O