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Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection

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Abstract

There has been a dramatic expansion of DNA sequence information compiled over the past several years for a variety of eukaryotic and prokaryotic genomes. Accompanying this increase in knowledge of genomic structure and organization has been a growing interest in studying the function of individual genes including regulation of their expression. A number of methods such as Northern blotting, ribonuclease protection assay, and hybridization arrays have been developed to analyze gene expression at the transcriptional (mRNA) level. Although quantitative estimates of mRNA transcripts can be obtained from each of these methods, oftentimes they lack sufficient sensitivity or the methodology is too costly or too labor-intensive to be applied to the analysis of a large number of samples. The most sensitive method for analyzing gene expression at the mRNA level involves the combination of reverse transcription and polymerase chain reaction (RT-PCR). However, in order to provide accurate quantitative estimates of gene expression, a rapid and efficient method is required for separation and detection of the double-stranded DNA (dsDNA) products of RT-PCR. Recent advances in capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) have made this method suitable for the automated analysis of large numbers of RT-PCR samples. An overview of the application of CE/LIF to quantitative analysis of gene expression by RT-PCR is presented along with selected protocols and examples. Both relative-quantitative (RQ) and quantitative-competitive (QC) approaches to RT-PCR are discussed in conjunction with the use of CE/LIF for rapid and accurate quantitative analysis of PCR products.

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References

  1. Bustin, S.A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endo. 25, 169–193.

    Article  CAS  Google Scholar 

  2. Jenkins, F.J. (1994) Basic methods for the detection of PCR products. PCR Meth. Appl. 4, S77-S82.

    Google Scholar 

  3. Righetti, P.G. and Gelfi, C. (1996) Capillary electrophoresis of DNA, in Capillary Electrophoresis in Analytical Biotechnology (Righetti, P.G., ed.), CRC Press Inc., Boca Raton, FL, pp. 431–476.

    Google Scholar 

  4. Ulfelder, K.J. and McCord, B.R. (1997) Separation of DNA by capillary electrophoresis, in Handbook of Capillary Electrophoresis, Second Edition (Landers, J.P., ed.), CRC Press Inc., Boca Raton, FL, pp. 347–378.

    Google Scholar 

  5. Butler, J.M. (1998) Separation of DNA restriction fragments and PCR products, in Analysis of Nucleic Acids by Capillary Electrophoresis (Heller, C. ed.), Verlag Vieweg, Wiesbaden, pp. 195–217.

    Google Scholar 

  6. Guttman, A. and Schwartz, H.E. (1998) Separation of DNA, in Capillary Electrophoresis Theory and Practice, Second Edition, (Camilleri, P., ed.), CRC Press, Boca Raton, FL, pp. 397–439.

    Google Scholar 

  7. Heller, C. (2001) Principles of DNA separation with capillary electrophoresis. Electrophoresis 22, 629–643.

    Article  PubMed  CAS  Google Scholar 

  8. Olivera, B.M., Baine, P. and Davidson, N. (1964) Electrophoresis of nucleic acids. Biopolymers 2, 245–257.

    Article  CAS  Google Scholar 

  9. Heller, C. (2001) Influence of polymer concentration and polymer composition on capillary electrophoresis of DNA, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 1: Introduction to the Capillary Electrophoresis of Nucleic Acids (Mitchelson, K.R., and Cheng, J., eds.), Humana, Totowa, NJ, pp. 111–123.

    Google Scholar 

  10. Williams, S.J. and Williams, P.M. (2001) Quantitation of mRNA by competitive PCR using capillary electrophoresis, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 2: Practical Applications of Capillary Electrophoresis (Mitchelson, K.R., and Cheng, J., eds.), Humana, Totowa, NJ, pp. 243–252.

    Google Scholar 

  11. Liu, M.-S. and Chen, F.-T.A. (2000) Rapid analysis of amplified double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection. Molec. Biotech. 15, 143–146.

    Article  CAS  Google Scholar 

  12. Huang, M.-F., Hsu, C.-E., Tseng, W.-L., Lin, Y.-C. and Chang, H.-T. (2001) Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions. Electrophoresis 22, 2281–2290.

    Article  PubMed  CAS  Google Scholar 

  13. Devaney, J.M. and Marino, M.A. (2001) Purification methods for preparing polymerase chain reaction products for capillary electrophoresis, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 1: Introduction to the Capillary Electrophoresis of Nucleic Acids (Mitchelson, K.R., and Cheng, J., eds.), Humana, Totowa, NJ, pp. 43–49.

    Google Scholar 

  14. Schwarz, H.E. and Ulfelder, K.J. (1992) Capillary electrophoresis with laser induced fluorescence detection of PCR fragments using thiazole orange. Anal. Chem. 64, 1737–1740.

    Article  Google Scholar 

  15. J. Skeidsvoll, J. and M. Ueland, M. (1995) Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I. Anal. Biochem. 231, 359–365.

    Article  PubMed  CAS  Google Scholar 

  16. McPherson, M.J. and Moller, S.G. (2000) Analysis of gene expression, in The Basics from Background to Bench: PCR (McPherson, M.J. and Moller, S.G., eds.), Bios Scientific Publishers LTD, Oxford, UK, pp. 183–211.

    Google Scholar 

  17. McPherson, M.J. and Moller, S.G. (2000) Understanding PCR, in The Basics from Background to Bench: PCR (McPherson, M.J. and Moller, S.G., eds.), Bios Scientific Publishers LTD, Oxford, UK, pp. 9–21.

    Google Scholar 

  18. Wenz, H.M., Dailey, D., and Johnson, M.D. (2001) Development of a high-throughput capillary electrophoresis protocol for DNA fragment analysis, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 2: Practical Applications of Capillary Electrophoresis (Mitchelson, K.R., and Cheng, J., eds.), Humana, Totowa, NJ, pp. 3–17.

    Google Scholar 

  19. Butler, J.M., McCord, B.R., Jung, J.M., Lee, J.A., Budowle, B. and Allen, R.O. (1995) Application of dual internal standards for precise sizing of polymerase chain reaction products using capillary electrophoresis. Electrophoresis 16, 974–980.

    Article  PubMed  CAS  Google Scholar 

  20. Saric, T. and Shain, S.A. (1997) Semiquantitative RT-PCR: Enhancement of assay accuracy and reproducibility. Biotechniques 22, 631–636.

    Google Scholar 

  21. Butler, J.M., Ruitberg, C.M. and Vallone, P.M. (2001) Capillary electrophoresis as a tool for optimization of multiplex PCR reactions. Fresenius J. Anal. Chem. 369, 200–205.

    Article  PubMed  CAS  Google Scholar 

  22. Zhong, H. and Simons, J.W. (1999) Direct comparison of GAPDH, β-actin, cyclophillin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem. Biophys Res. Comm. 259, 523–526.

    Article  PubMed  CAS  Google Scholar 

  23. Suzuki, T. Higgins, P.J. and Crawford, D.R. (2000) Control selection for RNA quantification. Biotechniques 29, 332–337.

    PubMed  CAS  Google Scholar 

  24. Richards, M.P. (1989) Recent developments in trace element metabolism and function: Role of metallothionein in copper and zinc metabolism. J. Nutr. 119, 1062–1070.

    PubMed  CAS  Google Scholar 

  25. Henegariu, O., Heerema, N.A., Dlouhy, S.R., Vance, G.H. and Vogt, P.H. (1997) Multiplex PCR: Critical parameters and Step-by-step protocol. Biotechniques 23, 504–511.

    PubMed  CAS  Google Scholar 

  26. Vehaskari, V.M., Hempe, J.M., Manning, J., Aviles, D.H. and Carmichael, M.C. (1998) Developmental regulation of ENaC subunit mRNA levels in rat kidney. Am. J. Physiol. 274 (Cell. Physiol. 43), C1661-C1666.

    PubMed  CAS  Google Scholar 

  27. Riedy, M.C., Timm, E.A. Jr. and Stewart, C.C. (1995) Quantitative RT-PCR for measuring gene expression. Biotechniques 18, 70–76.

    PubMed  CAS  Google Scholar 

  28. Souaze, F., Ntodou-Thome, A., Tran, C.Y., Rostene, W. and Forgez, P. (1996) Quantitative RT-PCR: Limits and accuracy. Biotechniques 21, 280–285.

    PubMed  CAS  Google Scholar 

  29. Auboeuf, D. and Vidal, H. (1997) The use of the reverse transcription-competitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples. Anal. Biochem. 245, 141–148.

    Article  PubMed  CAS  Google Scholar 

  30. O’Connell, J., Goode, T. and Shanahan, F. (1998) Quantitative measurement of mRNA expression by competitive RT-PCR, in Methods in Molecular Biology, vol. 92: PCR in Bioanalysis (Meltzer, S.J., ed), Humana, Totowa, NJ, pp. 183–193.

    Chapter  Google Scholar 

  31. Freeman, W.M., Walker, S.J. and Vrana, K.E. (1999) Quantitative RT-PCR: Pitfalls and potential. Biotechniques 26, 112–125.

    PubMed  CAS  Google Scholar 

  32. Vu, H. L., Troubetzkoy, S., Nguyen, H.H., Russell, M.W. and Mestecky, J. (2000) A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis. Nucleic Acids Res. 28, e18.

    Google Scholar 

  33. Richards, M.P., Ashwell, C.M. and McMurtry, J.P. (1999) Analysis of leptin gene expression in chickens using reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr. A 853, 321–335.

    Article  PubMed  CAS  Google Scholar 

  34. Richards, M.P., Ashwell, C.M. and McMurtry, J.P. (2000) Quantitative analysis of leptin mRNA using competitive reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis 21, 792–798.

    Article  PubMed  CAS  Google Scholar 

  35. Fasco, M., Treanor, C. P., Spivack, S., Figge, H. L. and Kaminsky, L. S. (1995) Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence. Anal. Biochem. 224, 140–147.

    Article  PubMed  CAS  Google Scholar 

  36. Borson, N. D., Strausbauch, M. A., Wettstein, P. J., Oda, R. P., Johnston, S. L. and Landers, J. P. (1998) Direct quantitation of RNA transcripts by competitive single-tube RT-PCR and capillary electrophoresis. Biotechniques 25, 130–137.

    PubMed  CAS  Google Scholar 

  37. Bor, M.V., Sorenson, B.S. and Nexo, E. (2000) Simultaneous quantitation of several mRNA species by calibrated reverse transcription polymerase chain reaction and capillary electrophoresis: Analysis of the epidermal growth factor receptor and its activating ligands EGF, TGF-a, and HB-EGF in rat liver. Lab. Invest. 80, 983–986.

    PubMed  CAS  Google Scholar 

  38. Reddy, N.R.J., Wilkie, B.N. and Mallard, B.A. (1996) Construction of an internal control to quantitate multiple porcine cytokine mRNAs by RT-PCR. Biotechniques 21, 868–875.

    PubMed  CAS  Google Scholar 

  39. Jensen, L.E. and Whitehead, A.S. (1998) Competitive reverse transcription polymerase chain reaction for quantifying pre-mRNA and mRNA of major acute phase proteins. J. Immunol. 215, 45–58.

    Article  CAS  Google Scholar 

  40. Wooley, A.T. and Mathies, R.A. (1994) Ultra-high-speed DNA fragment separation using microfabricated capillary array electrophoresis chips. Proc. Natl. Acad. Sci. USA 91, 11348–11352.

    Article  Google Scholar 

  41. Medintz, I.L., Paegel, B.M. and Mathies, R.A. (2001) Microfabricated capillary array electrophoresis DNA analysis systems. J. Chromatogr. A 924, 265–270.

    Article  PubMed  CAS  Google Scholar 

  42. Schmalzing, D., Koutny, L., Adourian, A., Chisholm, D., Matsudaira, P. and Ehrlich, D. (2001) Genotyping by microdevice electrophoresis, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 2: Practical Applications of Capillary Electrophoresis (Mitchelson, K.R. and Cheng, J., eds.), Humana, Totowa, NJ, pp. 163–173.

    Google Scholar 

  43. Xie, W., Yang, R., Xu, J., Zhang, L., Xing, W. and Cheng, J. (2001) Microchip-based capillary electrophoresis systems, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 1: Introduction to the Capillary Electrophoresis of Nucleic Acids (Mitchelson, K.R. and Cheng, J., eds.), Humana, Totowa, NJ, pp. 67–83.

    Google Scholar 

  44. Dovichi, N. and Zhang, J.Z. (2001) DNA sequencing by capillary array electrophoresis, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 1: Introduction to the Capillary Electrophoresis of Nucleic Acids (Mitchelson, K.R. and Cheng, J., eds.), Humana, Totowa, NJ, pp. 85–94.

    Google Scholar 

  45. Muller, O., Foret, F. and Karger, B.L. (1995) Design of a high precision fraction collector for capillary electrophoresis. Anal. Chem. 67, 2974–2980.

    Article  PubMed  CAS  Google Scholar 

  46. Muth, J., Muller, O., Berka, J., Leonard, J.T. and Karger, B.L. (1996) DNA sequence analysis of Prinker-modified restriction fragments after collection from capillary electrophoresis with replaceable matrices. J. Chromatogr. A 744, 303–310.

    Article  PubMed  CAS  Google Scholar 

  47. Lacher, N.A., Garrison, K.E., Martin, R.S. and Lunte, S.M. (2001) Microchip capillary electrophoresis/electrochemistry. Electrophoresis 22, 2526–2536.

    Article  PubMed  CAS  Google Scholar 

  48. Zhang, N., Tan, H. and Yeung, E.S. (1999) Automated and integrated system for high-throughput DNA genotyping directly from blood. Anal. Chem. 71, 1138–45.

    Article  PubMed  CAS  Google Scholar 

  49. Ferrance, J.P., Giordano, B. and Landers, J.P. (2001) Toward effective PCR-based amplification on microfabricated chips, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 2: Practical Applications of Capillary Electrophoresis (Mitchelson, K.R. and Cheng, J., eds.), Humana, Totowa, NJ, pp. 191–204.

    Google Scholar 

  50. Hong, J.W., Fujii, T., Seki, M., Yamamoto, T. and Endo, I. (2001) Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip. Electrophoresis 22, 328–333.

    Article  PubMed  CAS  Google Scholar 

  51. Jang, G. and Harrison, D.J. (2000) mRNA isolation in a microfluidic device for eventual integration of cDNA library construction. Analyst 125, 2176–2179.

    Article  Google Scholar 

  52. Burns, M.A., Johnson, B.N., Brahmasandra, S.N., Handique, K., Webster, J.R., Krishman, M., Sammarco, T.S., Man, P.M., Jones, D., Heldsinger, D., Mastrangelo, C.H. and Burke, D.T. (1998) An integrated nanoliter DNA analysis device. Science 282, 484–487.

    Article  PubMed  CAS  Google Scholar 

  53. Irie, T., Oshida, T., Hasegawa, H., Matsuoka, Y., Li, T., Oya, Y., Tanaka, T., Tsujimoto, G and Kambara, H. (2000) Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes. Electrophoresis 21, 367–374.

    Article  PubMed  CAS  Google Scholar 

  54. Zhao, X. and George, K.S. (2001) Differential display analysis by capillary electrophoresis, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 2: Practical Applications of Capillary Electrophoresis (Mitchelson, K.R. and Cheng, J., eds.), Humana, Totowa, NJ, pp. 259–267.

    Google Scholar 

  55. Stanta, G., Bonin, S. and Lugli, M. (2001) Quantitative RT-PCR from fixed paraffin-embedded tissues by capillary electrophoresis, in Methods in Molecular Biology, vol. 162: Capillary Electrophoresis of Nucleic Acids, vol. 2: Practical Applications of Capillary Electrophoresis (Mitchelson, K.R. and Cheng, J., eds.), Humana, Totowa, NJ, pp. 253–258.

    Google Scholar 

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  1. United States Department of Agriculture; Agricultural Research Service, Animal and Natural Resources Institute, Growth Biology Laboratory, 10300, Baltimore Avenue, Building 200, Room 206, BARC-East, 20705-2350, Beltsville, Maryland

    Mark P. Richards

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Richards, M.P., Poch, S.M. Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection. Mol Biotechnol 21, 19–37 (2002). https://doi.org/10.1385/MB:21:1:019

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