Skip to main content
Log in

Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli

  • Methods
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript


Real-time polymerase chain reaction (PCR)-based methodology for the determination of rRNA gene (rrn) copy number was introduced and demonstrated. Both absolute and relative quantifications were tested with Escherichia coli. The separate detection of rRNA gene and chromosomal DNA was achieved using two primer sets, specific for 16S rRNA gene and for D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. As dxs is a single-copy gene of E. coli chromosomal DNA, the rrn copy number can be determined as the copy ratio of rrn to dxs. This methodology was successfully applied to determine the rrn copy number in E. coli cells. The results from absolute and relative quantifications were identical and highly reproducible with coefficient of variation (CV) values of 1.8–4.6%. The estimated rrn copy numbers also corresponded to the previously reported value in E. coli (i.e., 7), indicating that the results were reliable. The methodology introduced in this study is faster and cost-effective without safety problems compared to the traditionally used Southern blot analysis. The fundamentals in our methodology would be applicable to any microorganism, as long as having the sequence information of the rRNA gene and another chromosomal gene with a known copy number.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2

Similar content being viewed by others


  • Blattner FR, Plunkett G III, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462

    Article  CAS  PubMed  Google Scholar 

  • Burgos JS, Ramirez C, Tenorio R, Sastre I, Bullido MJ (2002) Influence of reagents formulation on real-time PCR parameters. Mol Cell Probe 16:257–260

    Article  CAS  Google Scholar 

  • Candela M, Vitali B, Matteuzzi D, Brigidi P (2004) Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR. Lett Appl Microbiol 38:229–232

    Article  CAS  PubMed  Google Scholar 

  • Crosby LD, Criddle CS (2003) Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity. BioTechniques 34:2–9

    Article  Google Scholar 

  • Deprez RHL, Fijnvandraat AC, Ruijter JM, Moorman AFM (2002) Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 307:63–69

    Article  Google Scholar 

  • Ferre F (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods and Appl 2:1–9

    Article  CAS  Google Scholar 

  • Hahn F, Eubanks LM, Testa CA, Blagg BJ, Baker JA, Poulter CD (2001) 1-deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus. J Bacteriol 183:1–11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klappenbach JA, Saxman PR, Cole JR, Schmidt TM (2001) rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res 29:181–184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klatt CG, LaPara TM (2003) Aerobic biological treatment of synthetic municipal wastewater in membrane-coupled bioreactors. Biotechnol Bioeng 82:313–320

    Article  CAS  PubMed  Google Scholar 

  • Klein D (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 8:257–260

    Article  CAS  PubMed  Google Scholar 

  • LaPara TM, Nakatsu CH, Pantea L, Alleman JE (2000) Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Appl Environ Microbiol 66:3951–3959

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee C, Kim J, Shin SG, Hwang S (2006) Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123:273–280

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2-DDCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Nystorm K, Biller M, Grahn A, Lindh M, Larson G, Olofsson S (2004) Real time PCR for monitoring regulation of host gene expression in herpes simplex virus type 1-infected human diploid cells. J Virol Methods 118:83–94

    Article  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

  • Pushnova EA, Geier M, Zhu YS (2000) An easy and accurate agarose gel assay for quantitation of bacterial plasmid copy numbers. Anal Biochem 284:70–76

    Article  CAS  PubMed  Google Scholar 

  • Rasmussen R (2001) Quantification on the LightCycler. In: Meuer S, Wittwer C, Nakagawara K (eds) Rapid cycle real-time PCR: methods and applications. Springer, Berlin

    Google Scholar 

  • Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160

    Article  CAS  PubMed  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386

    CAS  PubMed  Google Scholar 

  • Ueno Y, Haruta S, Ishii M, Igarashi Y (2001) Changes in product formation an bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor. Appl Microbiol Biotechnol 57:65–73

    Article  CAS  PubMed  Google Scholar 

  • Vilalta A, Whitlow V, Martin T (2002) Real-time PCR determination of Escherichia coli genomic DNA contamination in plasmid preparations. Anal Biochem 301:151–153

    Article  CAS  PubMed  Google Scholar 

  • Whelan JA, Russel NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real time PCR. J Immunol Methods 278:261–269

    Article  CAS  PubMed  Google Scholar 

  • White D (2000) The physiology and biochemistry of prokaryotes, 2nd edn. Oxford University Press, Oxford

    Google Scholar 

  • Wilhelm J, Pingoud A, Hahn M (2003) Real-time PCR-based method for the estimation of genome sizes. Nucleic Acids Res 31:e56

    Article  PubMed  PubMed Central  Google Scholar 

  • Yu Y, Kim J, Hwang S (2006) Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures. Biotechnol Bioeng 93:424–433

    Article  CAS  PubMed  Google Scholar 

Download references


This work was supported in part by the Korea Ministry of Education (MOE) through the BK-21 program and by the Korea Science and Engineering Foundation (KOSEF) through the Advanced Environmental Biotechnology Research Center (R11-2003-006) at POSTECH. This research was also supported by New and Renewable Energy R&D program (2006-N-BI02-P-09) under the Korea Ministry of Commerce, Industry, and Energy (MOCIE).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Seokhwan Hwang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, C., Lee, S., Shin, S.G. et al. Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli . Appl Microbiol Biotechnol 78, 371–376 (2008).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: