(b) In PAXgene collector tubes for 2 h at room temperature
to allow for RNA stabilization, and then store at À20
C
until total RNA extraction.
4. Cut the skin open with a set of scissors and tweezers from the
throat to the rectum. Open the chest and peritoneal cavities
with a scalpel.
5. As for animals’ blood, any organ of interest can also be harvested, especially the liver and kidneys, and stored for further
analysis:
(a) For total DNA extraction, 25 mg samples should be
placed into MagNA Lyser Green Beads tubes containing
360 μL of lysis buffer from DNA extraction kit and stored
at 4
C until DNA extraction.
(b) For total RNA extraction, tissue samples (pieces of
~1 cm
3
) are placed in 1.5 mL RNAlater reagent for stabilization of total RNA at room temperature for 2 h before
conservation at À20
C until RNA extraction.
3.3 Leptospira
Burden Quantification
3.3.1 Total DNA
Extraction from Blood
1. Total DNA is extracted from blood samples kept in EDTA
collector tubes, using the QIAamp Blood Kit according to
manufacturer’s instructions.
2. After washing steps, the total DNA is eluted in elution buffer or
water.
3. Quantify
total
eluted
DNA
concentration
by
spectrophotometry.
Table 1
Blood and tissue sample storage conditions
Total DNA extraction
Total RNA extraction
Blood Storage
material
EDTA collector tubes
PAXgene collector tubes
Storage
temperature
4
C until DNA extraction, À20
C for
long-term storage
2 h at room temperature, followed by
long-term storage at À20
C until
total RNA extraction
Tissue Storage
material
25 mg samples into MagNA Lyser
green beads tubes containing 360 μL
of QIAamp lysis buffer
Tissue pieces (~1 cm
3 ) in 1.5 mL
RNAlater reagent
Storage
temperature
4
C until DNA extraction, À20
C for
long-term storage
2 h at room temperature, followed by
long-term storage at À20
C until
total RNA extraction
Summary of stabilization reagents and storage conditions followed for organs and blood collected from LiVV-infected
and uninfected control animals
Hamsters as a Model to Study Leptospirosis
249
to allow for RNA stabilization, and then store at À20
C
until total RNA extraction.
4. Cut the skin open with a set of scissors and tweezers from the
throat to the rectum. Open the chest and peritoneal cavities
with a scalpel.
5. As for animals’ blood, any organ of interest can also be harvested, especially the liver and kidneys, and stored for further
analysis:
(a) For total DNA extraction, 25 mg samples should be
placed into MagNA Lyser Green Beads tubes containing
360 μL of lysis buffer from DNA extraction kit and stored
at 4
C until DNA extraction.
(b) For total RNA extraction, tissue samples (pieces of
~1 cm
3
) are placed in 1.5 mL RNAlater reagent for stabilization of total RNA at room temperature for 2 h before
conservation at À20
C until RNA extraction.
3.3 Leptospira
Burden Quantification
3.3.1 Total DNA
Extraction from Blood
1. Total DNA is extracted from blood samples kept in EDTA
collector tubes, using the QIAamp Blood Kit according to
manufacturer’s instructions.
2. After washing steps, the total DNA is eluted in elution buffer or
water.
3. Quantify
total
eluted
DNA
concentration
by
spectrophotometry.
Table 1
Blood and tissue sample storage conditions
Total DNA extraction
Total RNA extraction
Blood Storage
material
EDTA collector tubes
PAXgene collector tubes
Storage
temperature
4
C until DNA extraction, À20
C for
long-term storage
2 h at room temperature, followed by
long-term storage at À20
C until
total RNA extraction
Tissue Storage
material
25 mg samples into MagNA Lyser
green beads tubes containing 360 μL
of QIAamp lysis buffer
Tissue pieces (~1 cm
3 ) in 1.5 mL
RNAlater reagent
Storage
temperature
4
C until DNA extraction, À20
C for
long-term storage
2 h at room temperature, followed by
long-term storage at À20
C until
total RNA extraction
Summary of stabilization reagents and storage conditions followed for organs and blood collected from LiVV-infected
and uninfected control animals
Hamsters as a Model to Study Leptospirosis
249