Molecular Techniques and Methods

Ribonuclease Protection Analysis
(RPA)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

  • Ribonuclease T1 (RNase T1) is a fungal endonuclease that cleaves single-stranded RNA after guanine residues, i.e., on their 3' end.
  • Ribonuclease A (RNase A) is an endonuclease that cleaves single-stranded RNA after cytidine and uridine, i.e., on their 3' end.



    MATERIALS AND SOLUTIONS

    5 M Ammonium Acetate (50 ml)
    Ammonium Acetate --------------------------------- 19.3 g
    DEPC-trated H2O to make a final vol. of -------- 50 ml
  • Since ammonium acetate decomposes by loss of ammonia, solutions should be prepared only from the pure salt which has been kept cool in a closed container.
  • Sterilize only by filtration; do not autoclave. Store solutions at 4oC.


    Hybridization Buffer (10 ml)
    80% Deionized Formamide ------------------ 8 ml
    40 mM PIPESpH6.4 ------------------------ 400 ul of 1 M PIPES
    400 mM NaCl ---------------------------- 800 ul of 5 M NaCl
    1 mM EDTA ------------------------------ 20 ul of 0.5 M EDTA
    DEPC-trated H2O -------------------------- 780 ul
  • Aliquote and keep at -20oC.


    RNase A/T1 Buffer (50 ml)
    10 mM Tris-HClpH7.5 ----------------------- 500 ul of 1 M Tris-HCl
    300 mM NaCl --------------------------------- 3 ml of 5 M NaCl
    5 mM EDTA ----------------------------------- 500 ul of 0.5 M EDTA
    Distilled H2O ----------------------------------- 46 ml


    Guanidine Thiocyanate RNase Inactivation Solution (75 ml)
    4 M Gluanidine thiocyanate ----------------- 36 g
    0.5% Sarkosyl ---------------------------- 3.75 ml of 10% Sarkosyl
    25 mM Na-citratepH7.0 --------------------- 18.75 ml of 1 M Na-citrate
    0.1 M 2-mercaptoethanol ------------------- 625 ul of 12 M 2-ME
    0.1% Antifoam (Sigma) --------------------- 75 ul
    Yeast RNA type III (10 ug/ul) ----------------- 750 ul
    DW ------------------------------------------ 15.8 ml
  • Keep at room temperature in dark place.


    Loading Buffer (1.07 ml)
    95% Deionized Formamide ---------------- 950 ul
    18 mM EDTA ----------------------------- 36 ul of 0.5 M EDTA
    0.025% SDS ----------------------------- 2.5 ul of 10% SDS
    0.2% BPB ------------------------------- 40 ul of 5% BPB
    0.2% Xylene cyanol --------------------- 40 ul of 5% Xylene cyanol
  • Keep at -20oC.


    Gel Fixative (200 ml)
    10 % Glacial Acetic Acid ---------------------------- 20 ml
    10 % Methanol -------------------------------------- 20 ml
    Distilled H2O ---------------------------------------- 160 ml




    PROCEDURES

    Antisense RNA Probe Preparation:

    1. After 20 ul of in vitro transcription, terminate the reaction by adding 2 ul of RNase-free DNase I.
  • Antisense RNA should have extra, nonspecific sequences (>60nt) which serves an indicator for positive RNase activity in RPA reaction.
  • IMPORTANT: For RNA equal loading optimization, test loading control RNA, such as GAPDH antisense probe, first.

    2. Mix by gentle flicking and quick spin in a microfuge. Incubate at 37oC for 30 minutes to diegst DNA template.

    3. 32P-labeled probe does not need to be gel prified.

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

    0.5M EDTA 1 ul
    Phenol:chloroform:isoamyl alcohol 100 ul
    Glycogen 10 ug
    5 M Ammounium acetate 100 ul
    DEPC-H2O 80 ul


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

    6. Do chloroform:isoamyl alcohol (50:1) extraction.

    7. Transfer the upper aqueous phase to a new tube and add 500 ul ice cold 100% Ethanol.

    8. Spin in a microfuge for 30 minutes at 4oC.

    9. Rinse pellet with 90% Et-OH to remove salt.

    10. Spin down for 5 min and air dry briefly.
  • Do not dry in a vacuum evaporator centrifuge.

    10. Add 120~300 ul of Hybridization Buffer (per 20 ul of in vitro transcipriotn reaction) and solubilize the pellet by vortexing.

    mRNA to detect Hybridization Buffer (ul)
    For medium abundant mRNAs (ex. GAPDH, Actin mRNAs) 120 ul
    For low abundant mRNAs (ex. c-fms mRNA) 300 ul

  • Excess antisense probes will be undigested and left as intact RNA.
  • Check specific activity by scintilation counter.
  • Proper RNA probe concentration. Use 2-8 x 104 cpm of a high specific activity probe per RPA reaction.
  • See 'Notes' below.


    RNA Preparation and Hybridization:
  • For the best results, use procedures that generate poly A+-mRNA of high quality and purity.

    11. Add the desired amount of target RNA (generally, 10 ug total RNA, or 0.1-1 ug poly A+mRNA) to 1.5 ml tubes.
  • Include a tube that contains 10 ug yeast tRNA as a RNase activity control.

    12. Precipitate RNA as follow.

    Total RNA or poly A+-mRNA 10 ul
    5 M Ammounium acetate 10 ul
    Glycogen 1 ul (10 ug)
    100% ET-OH 50 ul


    13. Spin in a microfuge for 30 minutes at 4oC.

    14. Rinse pellet with 90% Et-OH to remove salt.

    15. Spin down for 5 min and air dry briefly.

    16. Add 17 ul Hybridization Buffer to each sample.

    17. Vortex to dissolve.

    18. Dilute the probe with Hybridization Buffer to the appropriate concentration.

    19. Add 3 ul of diluted probe (2-8 x 104 cpm of a high specific activity probe per RPA reaction) to each 17 ul RNA sample and mix well.

    20. Place the samples in a heat block pre-warmed to 90oC for 3 min.

    21. Vortex vigorously and spin down at 4oC briefly.

    22. Place the samples in a heat block pre-warmed to 90oC for 1 min.

    23. Immediately transfer the samples in 40-42oC incubator and hybridize O/N (4-16h).
  • For c-fms RNA detection, hybridize at 41oC.
  • For GAPDH RNA detection, hybridize at 42oC.

    24. Remove samples and place at room temperature prior to the RNase treatments to allow the temperature to ramp down slowly.


    RNase Treatment:

    25. Add 200 ul RNase A/T1 solution containing 25 ug RNase A and 500 U RNase T1.
  • RNase amount should be determined experimentally, since each RNase has different activity units.

    26. Incubate at 37oC for 30min ~ 1h.
  • Signal intensity is different at different treatments. Over-digestion decreases signal.
  • For low abundant RNAs, incubate shorter time.

    27. Add, 20 ul of 10% Sarkosyl and 10 ul of 10ug/ul Proteinase K.
  • Sarkosyl helps proteinase reaction.

    28. Incubate at 37oC for 30 min.

    29. Add 250 ul Guanidine Thiocyanate RNase Inactivation Solution and 500 ul iPr-OH.

    30. Mix well and centrifuge for 30 min at 14,000 rpm, 4oC.

    31. Rinse pellet with 300 ul, 90% Et-OH.
  • This step is necessary to remove salts, which cause waving bands in the gel.

    32. Air dry briefly and add 10 ul of Loading Buffer.

    33. Denature at 90oC for 3 min.

    34. Transfer tubes in ice.

    35. Analyze in a denaturing Acrylamide/ 8M Urea gel at 185 volts, 40 min.
  • In this condition, load 0.05 ~ 0.1 ul undigested probe in 10 ul loading buffer as an undigested probe control.
  • For 50-450nt RNA separation, use 5% Acrylamide/8 M Urea Gel - Minigel/ 185 volts/ 35 min.
  • For 500-800nt RNA separation, use 3.5% Acrylamide/8 M Urea Gel.

    36. Optional: Fix the gel in the Gel Fixative for 15 min on one glass plate.
  • This procedure removes urea and decreases the time required for drying.

    37. Cut a piece of Whatmann 3MM paper to the appropriate size, and gently lay it on top of the gel.
  • Rub the back of the Whatmann paper with a paper towel, and then peel it away from the glass plate. The gel will remain stuck to the Whatmann paper.

    38. Cover the gel with cling film, smoothing out air bubbles with a paper towel.

    39. Dry the gel in a stab gel dryer at 80oC for 1 hr until the gel is dry.

    40. Remove the cling film, and autoradiograph the gel against a X-ray film in a cassette.




    NOTES

  • Consequences of Too Much Probe - Ambion

    One of the most common mistakes in designing a ribonuclease protection experiment is the addition of too much probe.
    RPA requires just enough to be in molar excess of the target mRNA since the hybridization is done in such a small volume (e.g. 20 µl).
    It is true that adding more probe will enhance the hybridization kinetics and give a stronger signal.
    In other words, more probe gives a better protected fragment signal in the same amount of time than a sample with a lesser amount of probe.
    However, if the problem is examined from an enzyme kinetics view, as more and more probe is added, the substrate concentration will eventually rise above the Vmax of the nuclease.
    This will result in a failure of the enzyme's ability to destroy all unprotected probe within the standard digestion incubation period.
    In order to prevent this, the probe concentration must be kept below the Vmax of the nuclease.
    Empirically, this is approximately no more than 1-2 fmol of probe per sample tube.
    This is acceptable, as at these concentrations the probe is still saturating in relation to target (except when detecting ribosomal RNA) for amounts of up to 80 µg of total RNA sample.
    In addition, most of the RNA found in a total RNA extract is ribosomal and transfer RNA, which does not serve as substrate for the nuclease and does not compete against mRNA for hydrolysis.
    Adding more nuclease is not a fix, because it tends to either overdigest the protected fragment during digestion or before loading a gel, and thus only contributes to higher background.

  • For moderately abundant messages (e.g. -actin or GAPDH), 2-8 x 104 cpm of a 300 base, high specific activity radiolabeled probe or 1 ng of a 300 base should be added per 10 g of total RNA.


    KIT INFORMATION



    REFERENCES
  • Michael Bordonaro, Colette F. Saccomanno, and Jeffrey L. Nordstrom, An Improved T1/A Ribonuclease Protection Assay, BioTechniques, 1994, 16, 3, 428-430.

  • Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. (1984) Efficient In Vitro Synthesis of Biologically Active RNA and RNA Hybridization Probes From Plasmids Containing a Bacteriophage SP6 Promoter. Nuc. Acids Res. 12: 7035-7056.

  • Winter, E., Yamamoto, F., Almognesa, C., Perucho, M. (1985) A Method to Detect and Characterize Point Mutations in Transcribed Genes: Amplification and Overexpression of the Mutant c-Ki-ras Allele in Human Tumor Cells. Proc. Nat. Acad. Sci. USA 82: 7575-7579.

  • Uchider, T. and Egami, F. (1967) The Specificity of Ribonuclease T2. J. of Biochem. 61: 44-49.

  • Ambion RPA


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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