BIO 631-60 Biotechnology: Gene Expression Spring 2002
Instructor: Dr. Henning Schneider
Laboratory Exercise # 5
Ribonuclease Protection Assay

Short Protocol

I. Introduction

A ribonuclease protection assay is an alternative to Nothern Hybridization and is based on the same principle. The expression of a gene (or the mRNA) can be detected by hybridizing a probe to isolated RNA. In contrast to a Northern hybridization, the hyrbridization of probe to RNA is carried out in solution and not on a membrane. Following the formation of a double-stranded RNA bewteen the probe and the RNA, single stranded RNA that did not hybridize with the probe and the labeled probe will be digested prior to gel electrophoresis, blotting and detection. Doubel stranded RNA will be protected from RNase digestion and cn be separated in a polyacrylamide gel. The RPA assay is somewhat similar to a S1 nuclease protection assay, but the RNase can be controlled better than S1 nuclease. In a RPA, mutliple probes can be used simultaneously to detect a number of mRNAs for an expression profile. In this exercise, we use the RPAII assay kit (Ambion) and the CDP-star detection method (Amersham Biosciences).

II. Materials and Methods

Solutions:

  • Hybridization Buffer III
  • RNase Digestion III buffer
  • yeast RNA ( 5mg/ml)
  • RNase Inactivatio/Precipitation III solution
  • gel loading buffer II
  • probe elution buffer
  • pTRI-Actin mouse vect
  • RNaseA/T1 mix (250 units/ml; 10,000 units/ml
  • RNase T1 (5,000U/ml)
  • ethanol (100%)
  • labeled RNA probe (Zebrafish 5-HT receptor, mouse-actin)
  • 0.5x TBE buffer
Equipment and supplies:
  • heat block/ waterbath 90°C
  • hybridization oven/ waterbath 42°C
  • RNase free sterile microcentrifuge tubes (1.5 ml)
  • mcirocentrifuge
  • pipettmans 20, 200, 1000 µl
  • RNase free filter tips
  • gel-box and powersupply for denaturing acrylamide gels
  • RNase free plastic pasteur pipettes

DAY 1

III. Hybridization

  1. setup three tubes: (1) mix 2.5 µg of total zebrafish/mouse RNA with 2.5 µl of labeled RNA probe, (2) mix 5 µg of total zebrafish/mouse RNA with 2.5 µl of labeled RNA probe, and (3) mix 2 µl of yeast RNA and 5 µl of RNA probe
  2. add 1/10 of the volume of 5M NH4OAc
  3. add 2.5 vol EtOH
  4. place tubes in -20°C freezer for 15 - 30 min
  5. centrifuge for 15 min at 12,000 rpm at 4°C
  6. remove supernatant
  7. air dry pellets for 5 min
  8. add 10 µl of hybridization buffer to each tube and vortex for 5 sec.
  9. quick spin in microcentrifuge tubes to collect contents in bottom of tubes
  10. incubate tubes for 3 min in heatblock (90-95°C) to denature samples
  11. incubate in hybridization oven at 42°C (minimum overnight)

DAY 2

IV. Ribonuclease digestion

  1. prepare three tubes: mix 150µl of RNase digestion III buffer with 1.5 µl RNase A/RNase T1
  2. prepare one tube with 150 µl RNase digestion II buffer w/o RNase A/RNase T1 (for yeast)
  3. remove tubes from hybridization oven an quick spin
  4. add diluted RNase digestion III solution
  5. incubate for 30 min at 37°C (in hybridization oven or waterbath)
  6. remove tubes from water bath or hybridization oven and quick spin
  7. to each tube, add 225 µl RNase inactivate/precipitation solution
  8. vortex for 5 sec and quick spin
  9. place tubes in -20°C freezer (min of 15 min)

  1. Acrylamide gel electrophoresis
  2. remove tubes and pin for 15 min at 12,000 xg at 4°C
  3. remove supernatant and prepare RNA for gel
  4. resuspend pellets in 4 - 10 µl gel loading buffer II
  5. prepare gel box with prepared 5% polyacrylamide gels and 1x TBE buffer
  6. release clamp assemblies from casting stand and assemble gel with cooling core
  7. pour running buffer into bottom of apparatus, insert cooling core at an angle, making sure no bubble are trapped underneath and
  8. pour running buffer into top
  9. apply sample with special long/flat tip pipette tips if necessary (5 -8 µl)
  10. run gel at 150 V constant voltage

V. Electroblotting

  1. equilibrate gel in transfer buffer 0.5 x TBE
  2. wet filter papers and fiber pads with 0.5x TBE
  3. fill buffer tank half way with buffer; put 1 inch stir bar in bottom, insert cooling unit
  4. open cassettes in a shallow dish, clear side on bottom (clear panel is anode side, gray is cathode)
  5. place we fiber pad on clear panel (centered)
  6. place wet filter paper on fiber pad
  7. then carefully place nitrocellulose on filter paper; layer with a little transfer buffer; avoid air bubbles
  8. cut nitrocellulose to exact size and notch lower right corner
  9. lay wet filter paper on top and a wet fiber pad
  10. close cassette carefully
  11. insert cassette so that gray panel faces gray side of apparatus, transfer while stirring for 30 min at 400 mA or 60 min at 200 mA
  12. after complete transfer, remove gel and membrane from stack and submerge them in transfer buffer,
  13. peel gel from the membrane and discard it
  14. rinse membrane briefly to remove acrylamide pieces
  15. crosslink at 1.2 mJ
  16. wrap membrane in saran wrap and store in -80°C freezer.

DAY 3

VI. Detection of probe using CDP-star method