Gene Expression Quantification from Pathogenic Bacterial Biofilms by Quantitative PCR

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Aug 22

PMID 37608108

Authors

Angela Franca

Nuno Cerca

Affiliations

Soon will be listed here.

Abstract

Quantitative PCR (qPCR) is one of the most used techniques to quantify gene expression in bacterial biofilms due to its easiness, sensitivity, and robustness. However, several practical aspects need to be considered to obtain accurate and reliable results. Here, we describe a detailed and optimized protocol to quantify mRNA transcripts from bacterial biofilms using qPCR, including pieces of advice to improve RNA quality, which ultimately increases the accuracy, consistency, and relevance of gene expression data.

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References

Franca A, Bento J, Cerca N . Variability of RNA quality extracted from biofilms of foodborne pathogens using different kits impacts mRNA quantification by qPCR. Curr Microbiol. 2012; 65(1):54-9. DOI: 10.1007/s00284-012-0124-5. View

Franca A, Melo L, Cerca N . Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis. BMC Res Notes. 2012; 4:572. PMC: 3260333. DOI: 10.1186/1756-0500-4-572. View

Franca A, Freitas A, Henriques A, Cerca N . Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol. PLoS One. 2012; 7(5):e37480. PMC: 3357405. DOI: 10.1371/journal.pone.0037480. View

Sieber M, Recknagel P, Glaser F, Witte O, Bauer M, Claus R . Substantial performance discrepancies among commercially available kits for reverse transcription quantitative polymerase chain reaction: a systematic comparative investigator-driven approach. Anal Biochem. 2010; 401(2):303-11. DOI: 10.1016/j.ab.2010.03.007. View

Sousa C, Franca A, Cerca N . Assessing and reducing sources of gene expression variability in Staphylococcus epidermidis biofilms. Biotechniques. 2014; 57(6):295-301. DOI: 10.2144/000114238. View

Carvalhais V, Delgado-Rastrollo M, Melo L, Cerca N . Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms. J Microbiol Methods. 2013; 95(2):195-200. DOI: 10.1016/j.mimet.2013.08.010. View

Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl M . Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett. 2006; 28(19):1601-13. DOI: 10.1007/s10529-006-9127-2. View

Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S . Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res. 2011; 39(9):e63. PMC: 3089491. DOI: 10.1093/nar/gkr065. View

Doma M, Parker R . RNA quality control in eukaryotes. Cell. 2007; 131(4):660-8. DOI: 10.1016/j.cell.2007.10.041. View

10.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

11.

Franca A, Carvalhais V, Maira-Litran T, Vilanova M, Cerca N, Pier G . Alterations in the Staphylococcus epidermidis biofilm transcriptome following interaction with whole human blood. Pathog Dis. 2014; 70(3):444-8. DOI: 10.1111/2049-632X.12130. View

12.

Carvalhais V, Franca A, Cerca F, Vitorino R, Pier G, Vilanova M . Dormancy within Staphylococcus epidermidis biofilms: a transcriptomic analysis by RNA-seq. Appl Microbiol Biotechnol. 2014; 98(6):2585-96. DOI: 10.1007/s00253-014-5548-3. View

13.

Magalhaes A, Franca A, Pereira M, Cerca N . Unveiling Co-Infection in Cystic Fibrosis Airways: Transcriptomic Analysis of and Dual-Species Biofilms. Front Genet. 2022; 13:883199. PMC: 9298864. DOI: 10.3389/fgene.2022.883199. View

14.

Magalhaes A, Franca A, Pereira M, Cerca N . RNA-based qPCR as a tool to quantify and to characterize dual-species biofilms. Sci Rep. 2019; 9(1):13639. PMC: 6754382. DOI: 10.1038/s41598-019-50094-3. View

15.

Sutton D, Conn G, Brown T, Lane A . The dependence of DNase I activity on the conformation of oligodeoxynucleotides. Biochem J. 1997; 321 ( Pt 2):481-6. PMC: 1218094. DOI: 10.1042/bj3210481. View

16.

Stahlberg A, Hakansson J, Xian X, Semb H, Kubista M . Properties of the reverse transcription reaction in mRNA quantification. Clin Chem. 2004; 50(3):509-15. DOI: 10.1373/clinchem.2003.026161. View

17.

Stahlberg A, Kubista M, Pfaffl M . Comparison of reverse transcriptases in gene expression analysis. Clin Chem. 2004; 50(9):1678-80. DOI: 10.1373/clinchem.2004.035469. View

18.

Abouhaidar M, Ivanov I . Non-enzymatic RNA hydrolysis promoted by the combined catalytic activity of buffers and magnesium ions. Z Naturforsch C J Biosci. 1999; 54(7-8):542-8. DOI: 10.1515/znc-1999-7-813. View

19.

Wilfinger W, Mackey K, Chomczynski P . Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997; 22(3):474-6, 478-81. DOI: 10.2144/97223st01. View

20.

Laurell H, Iacovoni J, Abot A, Svec D, Maoret J, Arnal J . Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime. Nucleic Acids Res. 2012; 40(7):e51. PMC: 3326333. DOI: 10.1093/nar/gkr1259. View