(including the kidney, liver, and brain), and are shed in the environment (soil and water) through the urine of infected hosts
[3]. Knowledge of the molecular basis of Leptospira pathogenicity
is very limited compared to other bacteria, mainly due to the lack of
genetic tools available for manipulation of leptospiral genome.
Inactivating a gene by allelic exchange in pathogenic Leptospira
strain is feasible but very inefficient. To study the function of a
given leptospiral gene, scientists usually rely on random transposon
insertion mutants [4, 5]. The transcriptomic approach is therefore
instrumental not only in identifying cellular pathways involved in
one particular physiological condition but also to speculate gene
function when mutants are not available. Effective RNA extraction
has allowed several laboratories to perform transcriptomic studies
in Leptospira, thereby leading to a better knowledge of bacterial
adaptation to host osmotic stress [6], in the presence of serum [7],
upon temperature changes [8, 9], and to the host environment
[10, 11].
Different methods can be used to extract RNA from a
biological sample. One method relies on the different solubilities
of cellular components in organic solvents and RNA precipitation
by alcohol. Another method is based on the ability of RNA to bind
to specific adsorbing material, such as silica and cellulose matrixes,
and is used in most commercial RNA purification kits. In a third
method, RNA is separated on density gradient centrifugation, but
this method is laborious and does not allow for simultaneous
processing of multiple samples.
Here, we describe the method based on RNA extraction with
an organic solvent and precipitation with alcohol currently applied
to Leptospira strains and allowing for high yields of pure and intact
RNA, compatible with the use of RNA-sequencing technology. In
this protocol, harvested Leptospira are first lysed in TRIzol™. This
reagent contains guanidinium isothiocyanate, a chaotropic agent
which is very effective at inactivating endogenous RNases. It also
contains low-pH phenol for separating DNA from RNA [12]. After
adding chloroform to the samples and subsequent centrifugation,
RNAs remain in the upper clear aqueous phase, while precipitated
proteins and DNA remain in the interphase and lower organic
phase, respectively. The RNA contained in the upper phase is
transferred to a new tube and undergoes alcohol precipitation.
The RNA pellet is then diluted in a suitable buffer. Traces of
contaminating DNA are eliminated by DNase treatment. This
guanidinium thiocyanate-phenol-chloroform extraction also
known as the “single-step method” greatly improved and expedited
RNA purification and has become the gold standard widely used for
any type of biological samples [13].
RNA quantification and purity can be determined by absorbance measurement at 260 and 280 nm. A ratio A 260 /A 280 of at
least 1.80 indicates an acceptable purity with low protein
42
Crispin Zavala-Alvarado and Nadia Benaroudj
[3]. Knowledge of the molecular basis of Leptospira pathogenicity
is very limited compared to other bacteria, mainly due to the lack of
genetic tools available for manipulation of leptospiral genome.
Inactivating a gene by allelic exchange in pathogenic Leptospira
strain is feasible but very inefficient. To study the function of a
given leptospiral gene, scientists usually rely on random transposon
insertion mutants [4, 5]. The transcriptomic approach is therefore
instrumental not only in identifying cellular pathways involved in
one particular physiological condition but also to speculate gene
function when mutants are not available. Effective RNA extraction
has allowed several laboratories to perform transcriptomic studies
in Leptospira, thereby leading to a better knowledge of bacterial
adaptation to host osmotic stress [6], in the presence of serum [7],
upon temperature changes [8, 9], and to the host environment
[10, 11].
Different methods can be used to extract RNA from a
biological sample. One method relies on the different solubilities
of cellular components in organic solvents and RNA precipitation
by alcohol. Another method is based on the ability of RNA to bind
to specific adsorbing material, such as silica and cellulose matrixes,
and is used in most commercial RNA purification kits. In a third
method, RNA is separated on density gradient centrifugation, but
this method is laborious and does not allow for simultaneous
processing of multiple samples.
Here, we describe the method based on RNA extraction with
an organic solvent and precipitation with alcohol currently applied
to Leptospira strains and allowing for high yields of pure and intact
RNA, compatible with the use of RNA-sequencing technology. In
this protocol, harvested Leptospira are first lysed in TRIzol™. This
reagent contains guanidinium isothiocyanate, a chaotropic agent
which is very effective at inactivating endogenous RNases. It also
contains low-pH phenol for separating DNA from RNA [12]. After
adding chloroform to the samples and subsequent centrifugation,
RNAs remain in the upper clear aqueous phase, while precipitated
proteins and DNA remain in the interphase and lower organic
phase, respectively. The RNA contained in the upper phase is
transferred to a new tube and undergoes alcohol precipitation.
The RNA pellet is then diluted in a suitable buffer. Traces of
contaminating DNA are eliminated by DNase treatment. This
guanidinium thiocyanate-phenol-chloroform extraction also
known as the “single-step method” greatly improved and expedited
RNA purification and has become the gold standard widely used for
any type of biological samples [13].
RNA quantification and purity can be determined by absorbance measurement at 260 and 280 nm. A ratio A 260 /A 280 of at
least 1.80 indicates an acceptable purity with low protein
42
Crispin Zavala-Alvarado and Nadia Benaroudj