used for the reaction. The manufacturer recommends using
5 μl of DNase Inactivating Reagent for 1 μl (2 U) of DNase.
23. It might be more comfortable to perform the DNase treatment
in a 0.5 ml polypropylene tube as it will ease removal of the
supernatant.
24. We recommend using the chip-based device of the Agilent
BioAnalyzer as its software is the only one allowing for a RIN
determination. This analysis is performed in transcriptomic
facilities.
Acknowledgments
Crispin Zavala-Alvarado is part of the Pasteur-Paris University
(PPU) International Ph.D. Program. Our laboratory has received
funding from the European Union’s Horizon 2020 research and
innovation programme under the Marie Sklodowska-Curie grant
agreement No. 665807 and from Fondation Etchebe `s-Fondation
de France. We would like to thank Robert Anthony Gaultney for his
suggestions on this manuscript.
References
1. Stears RL, Martinsky T, Schena M (2003)
Trends in microarray analysis. Nat Med 9
(1):140–145
2. Wang Z, Gerstein M, Snyder M (2009)
RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63
3. Picardeau M (2017) Virulence of the zoonotic
agent of leptospirosis: still terra incognita? Nat
Rev Microbiol 15:297–307
4. Bourhy P, Louvel H, Saint Girons I, Picardeau
M (2005) Random insertional mutagenesis of
Leptospira interrogans, the agent of Leptospirosis, using a mariner transposon. J Bacteriol
187(9):3255–3258
5. Murray GL, Morel V, Cerqueira GM, Croda J,
Srikram A, Henry R et al (2009) Genome-wide
transposon mutagenesis in pathogenic leptospira species. Infect Immun 77(2):810–816
6. Matsunaga J, Lo M, Bulach DM, Zuerner RL,
Adler B, Haake DA (2007) Response of Leptospira interrogans to physiologic osmolarity: relevance in signaling the environment-to-host
transition. Infect Immun 75(6):2864–2874
7. Patarakul K, Lo M, Adler B (2010) Global
transcriptomic response of Leptospira interrogans serovar Copenhageni upon exposure to
serum. BMC Microbiol 10(1):31–46
8. Qin J-H, Sheng Y-Y, Zhang Z-M, Shi Y-Z,
He P, Hu B-Y et al (2006) Genome-wide transcriptional analysis of temperature shift in
L. interrogans serovar lai strain 56601. BMC
Microbiol 6(1):51–60
9. Lo M, Bulach DM, Powell DR, Haake DA,
Matsunaga J, Paustian ML et al (2006) Effects
of temperature on gene expression patterns in
Leptospira interrogans serovar lai as assessed by
whole-genome microarrays. Infect Immun 74
(10):5848–5859
10. Xue F, Dong H, Wu J, Wu Z, Hu W, Sun A et al
(2010) Transcriptional responses of Leptospira
interrogans to host innate immunity: significant changes in metabolism, oxygen tolerance,
and outer membrane. PLoS Negl Trop Dis 4
(10):e857
11. Caimano MJ, Sivasankaran SK, Allard A,
Hurley D, Hokamp K, Grassmann AA et al
(2014) A model system for studying the transcriptomic and physiological changes associated with mammalian host-adaptation by
Leptospira interrogans serovar copenhageni.
PLoS Pathog 10(3):e1004004
12. Chomczynski P, Sacchi N (1987) Single-step
method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform
extraction.
Anal Biochem 162(1):156–159
50
Crispin Zavala-Alvarado and Nadia Benaroudj
5 μl of DNase Inactivating Reagent for 1 μl (2 U) of DNase.
23. It might be more comfortable to perform the DNase treatment
in a 0.5 ml polypropylene tube as it will ease removal of the
supernatant.
24. We recommend using the chip-based device of the Agilent
BioAnalyzer as its software is the only one allowing for a RIN
determination. This analysis is performed in transcriptomic
facilities.
Acknowledgments
Crispin Zavala-Alvarado is part of the Pasteur-Paris University
(PPU) International Ph.D. Program. Our laboratory has received
funding from the European Union’s Horizon 2020 research and
innovation programme under the Marie Sklodowska-Curie grant
agreement No. 665807 and from Fondation Etchebe `s-Fondation
de France. We would like to thank Robert Anthony Gaultney for his
suggestions on this manuscript.
References
1. Stears RL, Martinsky T, Schena M (2003)
Trends in microarray analysis. Nat Med 9
(1):140–145
2. Wang Z, Gerstein M, Snyder M (2009)
RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63
3. Picardeau M (2017) Virulence of the zoonotic
agent of leptospirosis: still terra incognita? Nat
Rev Microbiol 15:297–307
4. Bourhy P, Louvel H, Saint Girons I, Picardeau
M (2005) Random insertional mutagenesis of
Leptospira interrogans, the agent of Leptospirosis, using a mariner transposon. J Bacteriol
187(9):3255–3258
5. Murray GL, Morel V, Cerqueira GM, Croda J,
Srikram A, Henry R et al (2009) Genome-wide
transposon mutagenesis in pathogenic leptospira species. Infect Immun 77(2):810–816
6. Matsunaga J, Lo M, Bulach DM, Zuerner RL,
Adler B, Haake DA (2007) Response of Leptospira interrogans to physiologic osmolarity: relevance in signaling the environment-to-host
transition. Infect Immun 75(6):2864–2874
7. Patarakul K, Lo M, Adler B (2010) Global
transcriptomic response of Leptospira interrogans serovar Copenhageni upon exposure to
serum. BMC Microbiol 10(1):31–46
8. Qin J-H, Sheng Y-Y, Zhang Z-M, Shi Y-Z,
He P, Hu B-Y et al (2006) Genome-wide transcriptional analysis of temperature shift in
L. interrogans serovar lai strain 56601. BMC
Microbiol 6(1):51–60
9. Lo M, Bulach DM, Powell DR, Haake DA,
Matsunaga J, Paustian ML et al (2006) Effects
of temperature on gene expression patterns in
Leptospira interrogans serovar lai as assessed by
whole-genome microarrays. Infect Immun 74
(10):5848–5859
10. Xue F, Dong H, Wu J, Wu Z, Hu W, Sun A et al
(2010) Transcriptional responses of Leptospira
interrogans to host innate immunity: significant changes in metabolism, oxygen tolerance,
and outer membrane. PLoS Negl Trop Dis 4
(10):e857
11. Caimano MJ, Sivasankaran SK, Allard A,
Hurley D, Hokamp K, Grassmann AA et al
(2014) A model system for studying the transcriptomic and physiological changes associated with mammalian host-adaptation by
Leptospira interrogans serovar copenhageni.
PLoS Pathog 10(3):e1004004
12. Chomczynski P, Sacchi N (1987) Single-step
method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform
extraction.
Anal Biochem 162(1):156–159
50
Crispin Zavala-Alvarado and Nadia Benaroudj