New approach in RNA quantification using arginine-affinity chromatography: potential application in eukaryotic and chemically synthesized RNA


The knowledge of RNA’s role in biological systems and the recent recognition of its potential use as a reliable biotherapeutic tool increase the demand for development and validation of analytical methods for accurate analysis of RNA. Affinity chromatography is a unique technique because of the versatility of applications reliant on the affinity ligand used. Recently, an arginine-based matrix has been effectively applied in the purification of RNA because of the specific recognition mechanism for RNA molecules. This interaction is suggested to be due to the length of arginine side chain and its ability to produce good hydrogen bonding geometries, which promote multi-contact with RNA backbone or RNA bases, based on RNA folding. Thus, this work presents the development and validation of an analytical method with ultraviolet detection for the quantification of RNA using affinity chromatography with arginine amino acid as immobilized ligand. The method was validated according to International and European legislation for bioanalytical methods. The results revealed that the proposed method is suitable for the reliable detection, separation, and quantification of RNA, showing that the method is precise and accurate for concentrations up to 200 ng/μL of RNA. Furthermore, the versatility of the methodology was demonstrated by its applicability in the quantification of RNA from different eukaryotic cells and in crude samples of chemically synthesized RNA. Therefore, the proposed method demonstrates a potential multipurpose applicability in molecular biology RNA-based analysis and RNA therapeutics.

Proposed interactions occurring between arginine–agarose matrix and RNA molecules. Given the multiplicity of arginine side-chain interactions and depending upon RNA folding state, arginine will preferably bind to phosphate groups of RNA backbone or RNA bases.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Sharp PA (2009) The centrality of RNA. Cell 136(4):577–580

    Article  CAS  Google Scholar 

  2. 2.

    Kreiter S, Diken M, Selmi A, Tureci O, Sahin U (2011) Tumor vaccination using messenger RNA: prospects of a future therapy. Curr Opin Immunol 23(3):399–406

    Article  CAS  Google Scholar 

  3. 3.

    Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S, Hellemans J, Speleman F, Vandesompele J (2011) Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res 39(9):e63

    Article  CAS  Google Scholar 

  4. 4.

    Gjerde DT, Hoang L, Hornby D (2009) RNA purification and analysis: sample preparation, extraction, chromatography. Wiley, Weinheim

    Google Scholar 

  5. 5.

    Farrell RE (2012) RNA methodologies. In: Meyers RA (ed) Epigenetic regulation and epigenomics, vol 2, 1st. Wiley, Weinheim, pp 1–37

    Google Scholar 

  6. 6.

    Wieczorek D, Delauriere L, T. S (October 2012) Methods of RNA quality assessment. Promega Corporation Website. Accessed 20 Feb 2013

  7. 7.

    Heptinstall J, Rapley R (2000) Spectrophotometric analysis of nucleic acids. In: Rapley R (ed) The nucleic acid protocols handbook. Humana Press, Totowa, pp 57–60. doi:10.1385/1-59259-038-1:57

  8. 8.

    Barril P, Nates S (2012) Introduction to agarose and polyacrylamide gel electrophoresis matrices with respect to their detection sensitivities. In: Magdeldin DS (ed) Gel electrophoresis—principles and basics. InTech, Rijeka. doi:10.5772/38573

  9. 9.

    Ohta T, Tokishita S, Yamagata H (2001) Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli. Mutat Res 492(1–2):91–97

    CAS  Google Scholar 

  10. 10.

    Jones LJ, Yue ST, Cheung C-Y, Singer VL (1998) RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. Anal Biochem 265(2):368–374

    Article  CAS  Google Scholar 

  11. 11.

    Schmittgen TD, Lee EJ, Jiang J, Sarkar A, Yang L, Elton TS, Chen C (2008) Real-time PCR quantification of precursor and mature microRNA. Methods 44(1):31–38

    Article  CAS  Google Scholar 

  12. 12.

    Masotti A, Preckel T (2006) Analysis of small RNAs with the Agilent 2100 Bioanalyzer. Nat Methods. doi:10.1038/NMETH908

    Google Scholar 

  13. 13.

    Dickman MJ (2011) Ion pair reverse-phase chromatography: a versatile platform for the analysis of RNA. Chromatography Today 4:22

    Google Scholar 

  14. 14.

    McCarthy SM, Gilar M, Gebler J (2009) Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA. Anal Biochem 390(2):181–188

    Article  CAS  Google Scholar 

  15. 15.

    Azarani A, Hecker KH (2001) RNA analysis by ion-pair reversed-phase high performance liquid chromatography. Nucleic Acids Res 29(2):E7

    Article  CAS  Google Scholar 

  16. 16.

    Dickman MJ, Hornby DP (2006) Enrichment and analysis of RNA centered on ion pair reverse phase methodology. RNA 12(4):691–696

    Article  CAS  Google Scholar 

  17. 17.

    Waghmare SP, Pousinis P, Hornby DP, Dickman MJ (2009) Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions. J Chromatogr A 1216(9):1377–1382

    Article  CAS  Google Scholar 

  18. 18.

    Clonis YD (2006) Affinity chromatography matures as bioinformatic and combinatorial tools develop. J Chromatogr A 1101(1–2):1–24

    CAS  Google Scholar 

  19. 19.

    Magdeldin S, Moser A (2012) Affinity chromatography. In: Magdeldin DS (ed) Affinity chromatography: principles and applications. InTech, Rijeka. doi:10.5772/39087

  20. 20.

    Srisawat C, Goldstein IJ, Engelke DR (2001) Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures. Nucleic Acids Res 29(2):E4

    Article  CAS  Google Scholar 

  21. 21.

    Batey RT, Kieft JS (2007) Improved native affinity purification of RNA. RNA 13(8):1384–1389

    Article  CAS  Google Scholar 

  22. 22.

    Martins R, Queiroz JA, Sousa F (2010) A new affinity approach to isolate Escherichia coli 6S RNA with histidine-chromatography. J Mol Recognit 23(6):519–524

    Article  CAS  Google Scholar 

  23. 23.

    Martins R, Queiroz JA, Sousa F (2012) Histidine affinity chromatography-based methodology for the simultaneous isolation of Escherichia coli small and ribosomal RNA. Biomed Chromatogr 26(7):781–788

    Article  CAS  Google Scholar 

  24. 24.

    Martins R, Maia CJ, Queiroz JA, Sousa F (2012) A new strategy for RNA isolation from eukaryotic cells using arginine affinity chromatography. J Sep Sci 35(22):3217–3226

    Article  CAS  Google Scholar 

  25. 25.

    Sousa F, Cruz C, Queiroz JA (2010) Amino acids–nucleotides biomolecular recognition: from biological occurrence to affinity chromatography. J Mol Recognit 23(6):505–518

    Article  CAS  Google Scholar 

  26. 26.

    Treger M, Westhof E (2001) Statistical analysis of atomic contacts at RNA–protein interfaces. J Mol Recognit 14(4):199–214

    Article  CAS  Google Scholar 

  27. 27.

    Jeong E, Kim H, Lee SW, Han K (2003) Discovering the interaction propensities of amino acids and nucleotides from protein–RNA complexes. Mol Cells 16:161–167

    CAS  Google Scholar 

  28. 28.

    Yarus M, Widmann JJ, Knight R (2009) RNA–amino acid binding: a stereochemical era for the genetic code. J Mol Evol 69(5):406–429

    Article  CAS  Google Scholar 

  29. 29.

    U.S. Department of Health and Human Services (2001) Guidance for industry, bioanalytical method validation. UD Food and Drug Administration, Rockville

  30. 30.

    Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW (1979) Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Investig Urol 17(1):16–23

    CAS  Google Scholar 

  31. 31.

    Laurell H, Iacovoni JS, Abot A, Svec D, Maoret JJ, Arnal JF, Kubista M (2012) Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime. Nucleic Acids Res 40(7):e51

    Article  CAS  Google Scholar 

  32. 32.

    Fleige S, Pfaffl MW (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 27(2–3):126–139

    Article  CAS  Google Scholar 

  33. 33.

    Mo MH, Chen L, Yebo F, Wang W, Fu SW (2012) Cell-free circulating miRNA biomarkers in cancer. J Cancer 3:432–448

    Article  CAS  Google Scholar 

  34. 34.

    Kang K, Peng X, Luo J, Gou D (2012) Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling. J Anim Sci Biotechnol 3(1):4

    Article  CAS  Google Scholar 

  35. 35.

    Aranda R, Dineen SM, Craig RL, Guerrieri RA, Robertson JM (2009) Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration range. Anal Biochem 387(1):122–127

    Article  CAS  Google Scholar 

  36. 36.

    Pascolo S (2006) Vaccination with messenger RNA. Methods Mol Med 127:23–40

    CAS  Google Scholar 

  37. 37.

    Salem C, El-Alfy M, Leblond CP (1998) Changes in the rate of RNA synthesis during the cell cycle. Anat Rec 250(1):6–12

    Article  CAS  Google Scholar 

Download references


This work was supported by FCT, the Portuguese Foundation for Science and Technology (PTDC/EBB-BIO/114320/2009 and PEst-C/SAU/UI0709/2011 COMPETE). Rita Martins also acknowledges a fellowship (SFRH/BD/ 64100/2009) from FCT.

Conflict of interest

The authors declare no conflict of interest.

Author information



Corresponding author

Correspondence to F. Sousa.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Martins, R., Queiroz, J.A. & Sousa, F. New approach in RNA quantification using arginine-affinity chromatography: potential application in eukaryotic and chemically synthesized RNA. Anal Bioanal Chem 405, 8849–8858 (2013).

Download citation


  • Affinity
  • Arginine
  • Chromatography
  • RNA
  • Transcription