Assessment of fHbp Expression Level by Reverse Transcriptase Quantitative PCR and Promoter Sequence Analysis

  • Caroline Cayrou
  • Christopher D. BaylissEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1969)


Reverse transcriptase quantitative PCR (RT qPCR) is widely used for assessing the levels of expression of specific genes in various organisms. Here we describe a RT qPCR assay designed to determine the level of expression of fHbp in multiple isolates of Neisseria meningitidis. The level of expression is measured by a two-step qPCR and is associated with a promoter region analysis.

Key words

Gene expression fHbp qPCR Intergenic region Reverse transcription 


  1. 1.
    Seib KL, Serruto D, Oriente F, Delany I, Adu-Bobie J, Veggi D et al (2009) Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37. Infect Immun 77:292–299CrossRefGoogle Scholar
  2. 2.
    Madico G, Welsch JA, Lewis LA, McNaughton A, Perlman DH, Costello CE et al (2006) The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance. J Immunol 177:501–510CrossRefGoogle Scholar
  3. 3.
    Vernikos G, Medini D (2014) Bexsero(R) chronicle. Pathog Glob Health 108:305–316CrossRefGoogle Scholar
  4. 4.
    Gandhi A, Balmer P, York LJ (2016) Characteristics of a new meningococcal serogroup B vaccine, bivalent rLP2086 (MenB-FHbp; Trumenba(R)). Postgrad Med 128:548–556CrossRefGoogle Scholar
  5. 5.
    Cayrou C, Akinduko AA, Mirkes EM, Lucidarme J, Clark SA, Green LR et al (2018) Clustered intergenic region sequences as predictors of factor H binding protein expression patterns and for assessing Neisseria meningitidis strain coverage by meningococcal vaccines. PLoS One 13(5):e0197186CrossRefGoogle Scholar
  6. 6.
    Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 11:1026–1030Google Scholar
  7. 7.
    Sanders H, Brehony C, Maiden MC, Vipond C, Feavers IM (2012). The effect of iron availability on transcription of the Neisseria meningitidis fHbp gene varies among clonal complexes. Microbiology 158:869–876Google Scholar
  8. 8.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408CrossRefGoogle Scholar
  9. 9.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefGoogle Scholar
  10. 10.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Genetics and Genome BiologyUniversity of LeicesterLeicesterUK

Personalised recommendations