Fig. 2. In this analysis, the abundant 23S and 16S rRNAs are well
resolved, and the smaller peak corresponds to the 5S rRNA. Here, a
RIN value of 9.5 was obtained, which indicates pure and
non-degraded RNAs.
The yield of the purification method presented here can be up
to 75 μg of RNA per 10
9
Leptospira, and RIN values of at least 8.5
are routinely obtained, which makes RNA obtained suitable for
RNA-Seq analysis.
3.5 Storage
RNA can be stored at À20
C for a short-term storage but À80
C
is preferential for a long-term storage. RNA samples could be
aliquoted into several tubes to minimize freeze-thaw and reduce
RNase degradation occurring upon accidental RNase
contamination.
4 Notes
1. Only use autoclaved glassware dedicated for EMJH medium
preparation. In order to avoid contaminating the glassware
with components that could prevent growth of Leptospira, we
rinse beforehand the glassware with sterile WFI, and all the
chemical stock solutions are prepared with sterile WFI.
Fig. 2 Analysis of RNA preparation by capillary electrophoresis. 0.5 μg of total RNAs were analyzed on an
Agilent RNA 6000 Nano chip with the 2100 Bioanalyzer. (a) Capillary electrophoresis gel-like image of the
Agilent RNA 6000 ladder (lane M) and RNAs (lane RNAs). The migration position of 23S, 16S, and 5S rRNA is
indicated at the right of the image. (b) Electropherogram trace of the RNA preparation. The peaks
corresponding to different rRNAs are indicated. The RNA preparation displayed here has a RIN of 9.5. A
degraded RNA preparation would display a decrease in the 23S and 16S rRNA signal and a concomitant
increased baseline in the fast-migrating zone (before the position of the 16S rRNA peak)
Total RNA Extraction from Leptospira
47
resolved, and the smaller peak corresponds to the 5S rRNA. Here, a
RIN value of 9.5 was obtained, which indicates pure and
non-degraded RNAs.
The yield of the purification method presented here can be up
to 75 μg of RNA per 10
9
Leptospira, and RIN values of at least 8.5
are routinely obtained, which makes RNA obtained suitable for
RNA-Seq analysis.
3.5 Storage
RNA can be stored at À20
C for a short-term storage but À80
C
is preferential for a long-term storage. RNA samples could be
aliquoted into several tubes to minimize freeze-thaw and reduce
RNase degradation occurring upon accidental RNase
contamination.
4 Notes
1. Only use autoclaved glassware dedicated for EMJH medium
preparation. In order to avoid contaminating the glassware
with components that could prevent growth of Leptospira, we
rinse beforehand the glassware with sterile WFI, and all the
chemical stock solutions are prepared with sterile WFI.
Fig. 2 Analysis of RNA preparation by capillary electrophoresis. 0.5 μg of total RNAs were analyzed on an
Agilent RNA 6000 Nano chip with the 2100 Bioanalyzer. (a) Capillary electrophoresis gel-like image of the
Agilent RNA 6000 ladder (lane M) and RNAs (lane RNAs). The migration position of 23S, 16S, and 5S rRNA is
indicated at the right of the image. (b) Electropherogram trace of the RNA preparation. The peaks
corresponding to different rRNAs are indicated. The RNA preparation displayed here has a RIN of 9.5. A
degraded RNA preparation would display a decrease in the 23S and 16S rRNA signal and a concomitant
increased baseline in the fast-migrating zone (before the position of the 16S rRNA peak)
Total RNA Extraction from Leptospira
47