Molecular Techniques and Methods

Nonradioactive Labeling of RNA by In vitro Transcription

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In this procedure, DIG-UTP incorporates at approximately every 20-25th position in the transcripts. Since the nucleotide concentration does not become limiting in this reaction, 1 ug linear plasmid DNA (with a 1 kb insert) can produce approximately 10 ug of full-length labeled RNA transcript in a two hour incubation. Larger amounts of labeled RNA can be synthesized by scaling up the reaction components.




MATERIALS AND SOLUTIONS

10x RNA Labeling Mix (70 ul)
10 mM ATP ----------------------------- 7 ul of 100 mM ATP
10 mM CTP ----------------------------- 7 ul of 100 mM CTP
10 mM GTP ----------------------------- 7 ul of 100 mM GTP
6.5 mM UTP ----------------------------- 4.5 ul of 100 mM UTP
3.5 mM DIG-UTP ----------------------- 25 ul of 10 mM DIG-UTP
(or, 3.5 mM Biotin-UTP, or 3.5 mM Fluorescein-UTP)
DEPC-treated H2O ---------------------- 20 ul


10x Transcription Buffer (1 ml)
400 mM Tris-HCl (pH 8.0) ------------- 400 ul of 1 M Tris-HCl
60 mM MgCl2 -------------------------- 60 ul of 1 M MgCl2
100 mM DTT --------------------------- 100 ul of 1 M DTT
20 mM Spermidine ---------------------- 20 ul of 1 M Spermidine
DEPC-treated H2O --------------------- 420 ul





PROCEDURES

Template DNA Preparation

Preparation of linearized plasmid DNA:
  • The DNA to be transcribed should be cloned into the polylinker site of a transcription vector which contains a promoter for SP6, T7, or T3 RNA Polymerase.


  • To synthesize run-off transcripts, use a restriction enzyme that creates a 5'-overhang to linearize the template before transcription.


  • After the linearizing restriction digestion, do phenol: chloroform: IAA extraction and ethanol precipitation.


  • Resuspend the pellet in 10 mM Tris- HCl (pH 8.0).



  • Preparation of circular plasmid DNA:
  • The circular plasmid DNA can be used as template to create run-around transcripts.


  • Ethanol precipitate circular plasmid DNA.


  • Resuspend circular plasmid DNA in 10 mM Tris-HCl (pH 8.0).



  • Preparation of PCR fragment which has an RNA polymerase promoter ligated to its 5'-ends:
  • A PCR fragment that has the appropriate promoter ligated to its 5'-ends can serve as a transcription template.


  • Use polyacrylamide gel electrophoresis for purification of PCR fragments.


  • Elute PCR fragments in 10 mM Tris-HCl (pH 8.0).




  • In vitro Transcription

    1. Add the following components to a 1.5 ml microcentrifuge tube on ice.

    Linearized plasmid DNA 1 ug/ 2 ul
    10x RNA labeling mix 2 ul
    5x Transcription buffer 4 ul
    RNA polymerase (T7, SP6, T3)(20 U/ul) 2 ul
    RNase inhibitor 2ul
    DEPC H2O to make final vol. of 20 ul


    2. Incubate the tube for 2 hours at 37oC.

    3. To remove the template DNA, add 2 units DNase I (RNase- free) to the tube and incubate for 15 min at 37oC.

    4. Add 2 ul 0.2 M EDTA (pH 8.0) to the tube to stop the RNA polymerase reaction.

    5. To the reaction tube, add 2.5 ul 4 M LiCl and 75 ul prechilled (-20oC) 100% ethanol.

    6. Let the precipitate form for at least 30 minutes at -70oC (or, 2 hours at -20oC).

    7. Centrifuge the tube (at 13,000g) for 15 minutes at 4oC.

    8. Discard the supernatant.

    9. Wash the pellet with 50 ul ice-cold 70% (v/v) ethanol.

    10. Centrifuge the tube (at 13,000g) for 5 minutes at 4oC.

    11. Discard the supernatant.

    12. Air dry the pellet.

    13. Dissolve the RNA pellet in cold 100 ul DEPC-treated H2O.

    14. To estimate the yield of the transcript, do the following.

  • Run an aliquot (5 ul) of the transcript on an agarose gel (or, acrylamide gel) beside an RNA standard of known concentration.


  • Stain with ethidium bromide. Compare the relative intensity of staining between the labeled transcripts and the known standard.


  • Alternatively, transfer RNA to nylon membrane by blotting and detect DIG-labeled RNA by colorimetric methods.

    15. Dilute an aliquot of the probe solution to its working concentration (e.g., 0.2-10 ng/ul) in the hybridization buffer to be used for the in situ experiment.

  • (If you are not going to use the labeled probe immediately, store the probe solution at -70oC.)





  • NOTES

  • The amount of newly synthesized labeled RNA depends on the amount, size, site of linearization, and purity of the template DNA.


  • Instead of linearized plasmid DNA, 1 ug circular plasmid DNA or
    100-200 ng purified PCR fragment can be used as a template.


  • Longer incubations do not increase the yield of labeled RNA. To produce larger amounts of RNA, scale up the reaction components.


  • Since the amount of labeled RNA transcript is far in excess of the template DNA (by a factor of approx. 10), it is usually not necessary to remove the template DNA by DNase I treatment before an in situ hybridization experiment.


  • Caution - Avoid repeated freezing and thawing of the probe.





  • KIT INFORMATION




    REFERENCES

  • Dunn, J.J., and Studier, F.W., 1983, Complete nucleotides sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535.


  • Kassavetis, G.A., Butler, E.T., Roulland, D., and Chamberlin, M.J., 1982, Bacteriophage SP6-specific RNA polymerase. J. Biol. Chem. 257, 5779-5788.


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

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