contamination, suitable for RT-PCR. For performing RNA-Seq,
RNA preparation should be of the highest quality. The integrity of
RNA (i.e., absence of RNA degradation) can be assessed by analyzing the RNA preparation by capillary electrophoresis using, for
instance, the chip-based device of the Agilent BioAnalyzer. This
analysis will provide with a RIN (RNA integrity number) value that
represents an objective measurement of RNA integrity ranging
from 10 (highly intact RNA) to 1 (completely degraded RNA)
[14]. For RNA-Seq, a RIN value above 8 should be aimed.
The total RNAs obtained via this method are mostly ribosomal
RNAs. Depending on the analysis method used downstream,
depletion of ribosomal RNAs allowing enrichment of messenger
RNAs might be necessary.
2 Materials
We have applied this protocol to pathogenic Leptospira
(L. interrogans serovar Manilae strain L495) and saprophyte
(L. biflexa serovar Patoc strain Patoc) strains cultivated in vitro in
EMJH medium (see Note 1).
1. Albumin supplement: 10% (w/v) bovine serum albumin,
0.004% (w/v) zinc sulfate, 0.015% (w/v) magnesium chloride,
0.015% (w/v) calcium chloride, 0.1% (w/v) sodium pyruvate,
0.4% (w/v) glycerol, 1.25% (v/v) Tween 80, 0.0002% (w/v)
vitamin B12, 0.05% (w/v) ferrous sulfate (added at the last
moment) in sterile water for injection (WFI).
2. EMJH base: dissolve 2.3 g of Difco Leptospira Medium Base
EMJH (Becton Dickinson) in 900 ml sterile WFI. Autoclave
the solution.
3. EMJH medium: add 100 ml albumin supplement to 900 ml
EMJH base. Adjust the pH to 7.5 and filter sterilize the
solution.
4. Refrigerated centrifuge and rotor reaching 12,000 Â g.
5. Water bath at 55 and 4
C.
6. Vortexer.
7. Fume hood.
8. P1000, P200, P20, P10/2 micropipettes.
9. RNase-free barrier tips for pipettes.
10. 1–2 ml disposable serological plastic pipettes.
11. 1.5 ml RNase-free polypropylene microcentrifuge tubes (see
Note 2).
12. 50 and 15 ml RNase-free polypropylene conical tubes (see
Note 2).
Total RNA Extraction from Leptospira
43
RNA preparation should be of the highest quality. The integrity of
RNA (i.e., absence of RNA degradation) can be assessed by analyzing the RNA preparation by capillary electrophoresis using, for
instance, the chip-based device of the Agilent BioAnalyzer. This
analysis will provide with a RIN (RNA integrity number) value that
represents an objective measurement of RNA integrity ranging
from 10 (highly intact RNA) to 1 (completely degraded RNA)
[14]. For RNA-Seq, a RIN value above 8 should be aimed.
The total RNAs obtained via this method are mostly ribosomal
RNAs. Depending on the analysis method used downstream,
depletion of ribosomal RNAs allowing enrichment of messenger
RNAs might be necessary.
2 Materials
We have applied this protocol to pathogenic Leptospira
(L. interrogans serovar Manilae strain L495) and saprophyte
(L. biflexa serovar Patoc strain Patoc) strains cultivated in vitro in
EMJH medium (see Note 1).
1. Albumin supplement: 10% (w/v) bovine serum albumin,
0.004% (w/v) zinc sulfate, 0.015% (w/v) magnesium chloride,
0.015% (w/v) calcium chloride, 0.1% (w/v) sodium pyruvate,
0.4% (w/v) glycerol, 1.25% (v/v) Tween 80, 0.0002% (w/v)
vitamin B12, 0.05% (w/v) ferrous sulfate (added at the last
moment) in sterile water for injection (WFI).
2. EMJH base: dissolve 2.3 g of Difco Leptospira Medium Base
EMJH (Becton Dickinson) in 900 ml sterile WFI. Autoclave
the solution.
3. EMJH medium: add 100 ml albumin supplement to 900 ml
EMJH base. Adjust the pH to 7.5 and filter sterilize the
solution.
4. Refrigerated centrifuge and rotor reaching 12,000 Â g.
5. Water bath at 55 and 4
C.
6. Vortexer.
7. Fume hood.
8. P1000, P200, P20, P10/2 micropipettes.
9. RNase-free barrier tips for pipettes.
10. 1–2 ml disposable serological plastic pipettes.
11. 1.5 ml RNase-free polypropylene microcentrifuge tubes (see
Note 2).
12. 50 and 15 ml RNase-free polypropylene conical tubes (see
Note 2).
Total RNA Extraction from Leptospira
43