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Selection of the Internal Control Gene for Real-Time Quantitative RT-PCR Assays in Temperature Treated Leptospira

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Abstract

Analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. To avoid bias, real-time RT-PCR is referred to one or several internal control genes. Here, we sought to identify a gene to be used as normalizer by analyzing three functional distinct housekeeping genes (lipL41, flaB, and 16S rRNA) for their expression level and stability in temperature treated Leptospira cultures. Leptospira interrogans serovar Hardjo subtype Hardjoprajitno was cultured at 30°C for 7 days until a density of 106 cells/ml was reached and then shifted to physiological temperatures (37°C and 42°C) and to environmental temperatures (25°C and 30°C) during a 24 h period. cDNA was amplified by quantitative PCR using SYBR Green I technology and gene-specific primers for lipL41, flaB, and 16S rRNA. Expression stability (M) was assessed by geNorm and Normfinder v.18. 16S rRNA registered an average expression stability of M = 1.1816, followed by flaB (M = 1.682) and lipL41 (M = 1.763). 16S rRNA was identified as the most stable gene and can be used as a normalizer, as it showed greater expression stability than lipL41 and flaB in the four temperature treatments. Hence, comparisons of gene expression will have a higher sensitivity and specificity.

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Acknowledgments

This work was partially supported by Universidad Nacional Autónoma de México Grant IN222806 and UCMEXUS-CONACYT Grant CN0254. The authors acknowledge the Unidad de Síntesis del Instituto de Biotecnología de la Universidad Nacional Autónoma de México (Cuernavaca, Morelos, México) for the oligonucleotide synthesis. We thank Fausto Sánchez for his assistance and helpful suggestions on the use of the RT-PCR equipment and acknowledge the technical assistance of Pablo Vera.

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Authors and Affiliations

  1. Departamento de Microbiología e Inmunología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Coyoacán, 04510, México

    Erika Margarita Carrillo-Casas, Rigoberto Hernández-Castro, Francisco Suárez-Güemes & Alejandro de la Peña-Moctezuma

  2. Dirección de Investigación, Hospital General “Dr. Manuel Gea González”, Tlalpan, 14080, México

    Rigoberto Hernández-Castro

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  1. Erika Margarita Carrillo-Casas
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  2. Rigoberto Hernández-Castro
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  3. Francisco Suárez-Güemes
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  4. Alejandro de la Peña-Moctezuma
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Correspondence to Alejandro de la Peña-Moctezuma.

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Carrillo-Casas, E.M., Hernández-Castro, R., Suárez-Güemes, F. et al. Selection of the Internal Control Gene for Real-Time Quantitative RT-PCR Assays in Temperature Treated Leptospira . Curr Microbiol 56, 539–546 (2008). https://doi.org/10.1007/s00284-008-9096-x

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