Chapter 5
The Single-Step Method of RNA Purification Applied
to Leptospira
Crispin Zavala-Alvarado and Nadia Benaroudj
Abstract
Establishing a rapid method to obtain pure and intact RNA molecules has revolutionized the field of RNA
biology, enabling laboratories to routinely perform RNA analysis such as Northern blot, reverse transcriptase quantitative PCR, and RNA sequencing. Here, we describe an application of the effective single-step
method of RNA extraction (or guanidinium thiocyanate-phenol-chloroform extraction) applied to Leptospira species. This method is based on the powerful ability of guanidinium thiocyanate to inactivate RNases
and on the different solubilities of RNA and DNA in acidic phenol. This method allows one to reproducibly
obtain total RNAs with high yield and integrity, as determined by capillary electrophoresis, suitable for the
RNA sequencing technology.
Key words Spirochetes, Leptospira, RNA, Guanidinium thiocyanate, Phenol-chloroform extraction,
RIN, RNA-Seq, RT-PCR
1 Introduction
Efficient acquisition of pure and intact RNA molecule is a prerequisite for numerous analytical techniques such as reverse transcriptase quantitative PCR (RT-qPCR), Northern blotting, microarray
analysis, and RNA sequencing (RNA-Seq). Particularly powerful
are RNA-Seq technologies that allow for profiling and quantification of RNA. Knowing which gene is expressed and how genes are
regulated in a particular condition provides scientists with a comprehensive knowledge of the physiological state of cells. The pioneering transcriptomic studies performed in the 1990s have used
hybridization-based microarray technology [1]. Since the development of affordable, high-throughput sequencing technologies,
transcriptomes are determined by RNA-Seq [2].
Leptospira spp. are microorganisms with remarkable adaptation
capacities allowing survival in different ecological niches. Pathogenic strains disseminate in the blood of infected hosts, can persist
intracellularly in macrophages, colonize different animal tissues
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_5, © Springer Science+Business Media, LLC, part of Springer Nature 2020
41
The Single-Step Method of RNA Purification Applied
to Leptospira
Crispin Zavala-Alvarado and Nadia Benaroudj
Abstract
Establishing a rapid method to obtain pure and intact RNA molecules has revolutionized the field of RNA
biology, enabling laboratories to routinely perform RNA analysis such as Northern blot, reverse transcriptase quantitative PCR, and RNA sequencing. Here, we describe an application of the effective single-step
method of RNA extraction (or guanidinium thiocyanate-phenol-chloroform extraction) applied to Leptospira species. This method is based on the powerful ability of guanidinium thiocyanate to inactivate RNases
and on the different solubilities of RNA and DNA in acidic phenol. This method allows one to reproducibly
obtain total RNAs with high yield and integrity, as determined by capillary electrophoresis, suitable for the
RNA sequencing technology.
Key words Spirochetes, Leptospira, RNA, Guanidinium thiocyanate, Phenol-chloroform extraction,
RIN, RNA-Seq, RT-PCR
1 Introduction
Efficient acquisition of pure and intact RNA molecule is a prerequisite for numerous analytical techniques such as reverse transcriptase quantitative PCR (RT-qPCR), Northern blotting, microarray
analysis, and RNA sequencing (RNA-Seq). Particularly powerful
are RNA-Seq technologies that allow for profiling and quantification of RNA. Knowing which gene is expressed and how genes are
regulated in a particular condition provides scientists with a comprehensive knowledge of the physiological state of cells. The pioneering transcriptomic studies performed in the 1990s have used
hybridization-based microarray technology [1]. Since the development of affordable, high-throughput sequencing technologies,
transcriptomes are determined by RNA-Seq [2].
Leptospira spp. are microorganisms with remarkable adaptation
capacities allowing survival in different ecological niches. Pathogenic strains disseminate in the blood of infected hosts, can persist
intracellularly in macrophages, colonize different animal tissues
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_5, © Springer Science+Business Media, LLC, part of Springer Nature 2020
41