3.1 Cell Lysis
Optimally, the starting material should be in vitro-cultured Leptospira consisting of at least 10
9 cells. This corresponds to a 30 ml
Leptospira culture at exponential phase (see Note 8).
1. Centrifuge the Leptospira cells in a 50 ml conical tube for
15 min at 3000 Â g at 4
C (see Note 9).
2. Resuspend the cell pellet in 1 ml of TRIzol™ reagent and
transfer the suspension in a 1.5 ml polypropylene tube (see
Note 10).
3. Vortex well to fully resuspend the pellet.
4. Flash freeze samples in liquid nitrogen and store them at
À80
C until further use (see Note 11).
3.2 RNA Extraction
1. Thaw the sample(s) at room temperature (see Note 12).
2. Add 260 μl chloroform, mix thoroughly by inversion for 15 s,
and incubate for 10 min (see Note 13).
3. Centrifuge for 15 min at 12,000 Â g at 4
C. After the centrifugation, three phases are observed in the tube. The top clear
aqueous phase contains RNA, the white ring at the interphase
contains denatured precipitated proteins, and the bottom pink
organic phase contains DNA.
4. Carefully, transfer the aqueous top layer containing RNA to a
new clean 1.5 ml polypropylene tube (see Note 14).
5. Add 600 μl isopropanol to precipitate RNA. Mix thoroughly by
gently inverting the tube. Incubate for 5–10 min at room
temperature (see Note 15).
6. Centrifuge for 10 min at 12,000 Â g at 4
C and discard the
supernatant (see Note 16).
7. Wash the RNA pellet by adding 1 ml of 75% ethanol (see Note
17).
8. Centrifuge for 5 min at 12,000 Â g at 4
C. Discard the
supernatant (see Note 18).
9. Air-dry the RNA (see Note 19).
10. Resuspend the pellet in 40 μl RNase-free H 2 O by pipetting up
and down several times.
11. Incubate for 10 min in a water bath at 55
C in order to
enhance the resuspension of the pellet (see Note 20).
3.3 DNase Treatment
Here, we describe the DNase treatment using the Turbo DNAfree™ kit, but any other commercially available kit might work as
well.
1. Add 5 μl of the 10Â Turbo DNA-free™ buffer and 4 μl of
RNase-free H 2 O (provided in the kit) to 40 μl of the RNA
suspension obtained in step 11 in Subheading 3.2.
Total RNA Extraction from Leptospira
45
Optimally, the starting material should be in vitro-cultured Leptospira consisting of at least 10
9 cells. This corresponds to a 30 ml
Leptospira culture at exponential phase (see Note 8).
1. Centrifuge the Leptospira cells in a 50 ml conical tube for
15 min at 3000 Â g at 4
C (see Note 9).
2. Resuspend the cell pellet in 1 ml of TRIzol™ reagent and
transfer the suspension in a 1.5 ml polypropylene tube (see
Note 10).
3. Vortex well to fully resuspend the pellet.
4. Flash freeze samples in liquid nitrogen and store them at
À80
C until further use (see Note 11).
3.2 RNA Extraction
1. Thaw the sample(s) at room temperature (see Note 12).
2. Add 260 μl chloroform, mix thoroughly by inversion for 15 s,
and incubate for 10 min (see Note 13).
3. Centrifuge for 15 min at 12,000 Â g at 4
C. After the centrifugation, three phases are observed in the tube. The top clear
aqueous phase contains RNA, the white ring at the interphase
contains denatured precipitated proteins, and the bottom pink
organic phase contains DNA.
4. Carefully, transfer the aqueous top layer containing RNA to a
new clean 1.5 ml polypropylene tube (see Note 14).
5. Add 600 μl isopropanol to precipitate RNA. Mix thoroughly by
gently inverting the tube. Incubate for 5–10 min at room
temperature (see Note 15).
6. Centrifuge for 10 min at 12,000 Â g at 4
C and discard the
supernatant (see Note 16).
7. Wash the RNA pellet by adding 1 ml of 75% ethanol (see Note
17).
8. Centrifuge for 5 min at 12,000 Â g at 4
C. Discard the
supernatant (see Note 18).
9. Air-dry the RNA (see Note 19).
10. Resuspend the pellet in 40 μl RNase-free H 2 O by pipetting up
and down several times.
11. Incubate for 10 min in a water bath at 55
C in order to
enhance the resuspension of the pellet (see Note 20).
3.3 DNase Treatment
Here, we describe the DNase treatment using the Turbo DNAfree™ kit, but any other commercially available kit might work as
well.
1. Add 5 μl of the 10Â Turbo DNA-free™ buffer and 4 μl of
RNase-free H 2 O (provided in the kit) to 40 μl of the RNA
suspension obtained in step 11 in Subheading 3.2.
Total RNA Extraction from Leptospira
45