Purification and characterization of transcribed RNAs using gel filtration chromatography

Article metrics


RNA synthesis using in vitro transcription by phage T7 RNA polymerase allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. Previous purification approaches relied on gel electrophoretic or gravity-flow chromatography methods. We present here a protocol for the in vitro transcription of RNAs and subsequent purification using fast-performance liquid chromatography. This protocol greatly facilitates production of RNA in a single day from transcription to purification.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Schematic outline of RNA sample preparation.
Figure 2: Sample elution profiles obtained from the gel filtration chromatography step, demonstrating the separation of in vitro-transcribed RNA (10-ml reaction) from plasmid and nucleotide triphosphates (NTPs).
Figure 3: Example of a monomer/dimer equilibrium after an in vitro transcription (10-ml reaction).
Figure 4: RNA sample purity.


  1. 1

    Milligan, J.F., Groebe, D.R., Witherell, G.W. & Uhlenbeck, O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).

  2. 2

    Puglisi, J.D. & Wyatt, J.R. Biochemical and NMR studies of RNA conformation with an emphasis on RNA pseudoknots. Methods Enzymol. 261, 323–350 (1995).

  3. 3

    Kim, I., McKenna, S.A., Puglisi, E. & Puglisi, J.D. Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA 13, 289–294 (2007).

  4. 4

    Lukavsky, P.J. & Puglisi, J.D. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10, 889–893 (2004).

  5. 5

    Kim, I., Lukavsky, P.J. & Puglisi, J.D. NMR study of 100 kDa HCV IRES RNA using segmental isotope labeling. J. Am. Chem. Soc. 124, 9338–9339 (2002).

  6. 6

    Lukavsky, P.J., Kim, I., Otto, G.A. & Puglisi, J.D. Structure of HCV IRES domain II determined by NMR. Nat. Struct. Biol. 10, 1033–1038 (2003).

  7. 7

    Lukavsky, P.J., Otto, G.A., Lancaster, A.M., Sarnow, P. & Puglisi, J.D. Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function. Nat. Struct. Biol. 7, 1105–1110 (2000).

  8. 8

    Egger, D., Bolten, R., Rahner, C. & Bienz, K. Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization. Histochem. Cell Biol. 111, 319–324 (1999).

  9. 9

    Hanna, M.M., Yuriev, E., Zhang, J. & Riggs, D.L. Probing the environment of nascent RNA in Escherichia coli transcription elongation complexes utilizing a new fluorescent ribonucleotide analog. Nucleic Acids Res. 27, 1369–1376 (1999).

  10. 10

    Srivatsan, S.G. & Tor, Y. Fluorescent pyrimidine ribonucleotide: synthesis, enzymatic incorporation, and utilization. J. Am. Chem. Soc. 129, 2044–2053 (2007).

  11. 11

    Gong, P. & Martin, C.T. Mechanism of instability in abortive cycling by T7 RNA polymerase. J. Biol. Chem. 281, 23533–23544 (2006).

  12. 12

    Schenborn, E.T. & Mierendorf, R.C. Jr. A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res. 13, 6223–6236 (1985).

  13. 13

    Wyatt, J.R., Chastain, M. & Puglisi, J.D. Synthesis and purification of large amounts of RNA oligonucleotides. Biotechniques 11, 764–769 (1991).

  14. 14

    Cheong, H.K., Hwang, E., Lee, C., Choi, B.S. & Cheong, C. Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography. Nucleic Acids Res. 32, e84 (2004).

  15. 15

    Kieft, J.S. & Batey, R.T. A general method for rapid and nondenaturing purification of RNAs. RNA 10, 988–995 (2004).

  16. 16

    McKenna, S.A. et al. Molecular framework for the activaiton of PKR 282, 11474–11486 (2007).

  17. 17

    McKenna, S.A., Lindhout, D.A., Shimoike, T., Aitken, C.E. & Puglisi, J.D. Viral dsRNA inhibitors prevent self-association and autophosphorylation of PKR. J. Mol. Biol. 372, 103–113 (2007).

  18. 18

    McKenna, S.A., Kim, I., Liu, C.W. & Puglisi, J.D. Uncoupling of RNA binding and PKR kinase activation by viral inhibitor RNAs. J. Mol. Biol. 358, 1270–1285 (2006).

  19. 19

    Kim, I., Liu, C.W. & Puglisi, J.D. Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR. J. Mol. Biol. 358, 430–442 (2006).

  20. 20

    Draper, D.E., White, S.A. & Kean, J.M. Preparation of specific ribosomal RNA fragments. Methods Enzymol. 164, 221–237 (1988).

  21. 21

    Pleiss, J.A., Derrick, M.L. & Uhlenbeck, O.C. T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates. RNA 4, 1313–1317 (1998).

  22. 22

    Ferre-D'Amare, A.R. & Doudna, J.A. Use of cis- and trans-ribozymes to remove 5′ and 3′ heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res. 24, 977–978 (1996).

  23. 23

    Price, S.R., Ito, N., Oubridge, C., Avis, J.M. & Nagai, K. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J. Mol. Biol. 249, 398–408 (1995).

  24. 24

    Tzakos, A.G., Easton, L.E. & Lukavsky, P.J. Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies. Nat. Protoc. 2, 2139–2147 (2007).

  25. 25

    Gallo, S., Furler, M. & Sigel, R. In vitro transcription and purification of RNAs of different size. Chimia 59, 812–816 (2005).

  26. 26

    Walker, S.C., Avis, J.M. & Conn, G.L. General plasmids for producing RNA in vitro transcripts with homogeneous ends. Nucleic Acids Res. 31, e82 (2003).

  27. 27

    Collins, R.A. The Neurospora Varkud satellite ribozyme. Biochem. Soc. Trans. 30, 1122–1126 (2002).

  28. 28

    Davanloo, P., Rosenberg, A.H., Dunn, J.J. & Studier, F.W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039 (1984).

  29. 30

    Hames, B.D. & Rickwood, D. (eds). Gel Electrophoresis of Proteins. Oxford University Press, New York (1981).

  30. 29

    Puglisi, J.D. & Tinoco, I. Jr. Absorbance melting curves of RNA. Methods Enzymol. 180, 304–325 (1989).

  31. 31

    Pohl, T. Concentration of proteins and removal of solutes. Methods Enzymol. 182, 68–83 (1990).

  32. 32

    McPhie, P. Dialysis. Methods Enzymol. 22, 23–32 (1990).

Download references

Author information

Correspondence to Joseph D Puglisi.

Rights and permissions

Reprints and Permissions

About this article

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.