3. Results are then expressed as the number of Leptospira genome
equivalents per μg of DNA extracted from tissue samples or
blood (see Note 12).
3.4 Cytokine Gene
Expression Analysis
All steps should be carried out using a refrigerate block or on ice to
avoid RNA degradation, except otherwise cited.
3.4.1 Total RNA
Extraction from Blood
1. Total RNA is purified from blood collected as described in
Subheading 3.2 and stored in PAXgene collector tubes using
the PAXgene blood RNA system according to manufacturer’s
instructions.
2. Following the washing steps, RNA is eluted in 80 μL elution
buffer, and 40 μL of RNA eluate is treated for 30 min at 37
C
with TURBO DNase (2 units) for elimination of residual
genomic DNA.
3. Incubate the DNase-treated RNA at 65
C for 10 min for
complete secondary and tertiary structure denaturation.
4. Before long-term storage at À80
C, purified RNA is quantified by spectrophotometry through measurement of the optical
density (OD) at 260 nm.
3.4.2 Total RNA
Extraction from Organs
1. Prior to RNA extraction from organs collected as advised in
Subheading 3.2, tissue samples kept in RNAlater reagent are
first disrupted and homogenized using the MagNA Lyser
Instrument during 50 s at 4000 Â g and then collected after
centrifugation for 2 min at 18,000 Â g.
2. Total RNA is extracted from tissue lysates using the High Pure
tissue RNA Isolation Kit.
3. Following the washing steps, RNA is eluted in 80 μL elution
buffer, and 40 μL of RNA eluate is treated for 30 min at 37
C
with TURBO DNase (2 units) for elimination of residual
genomic DNA.
4. Incubate the DNase-treated RNA at 65
C for 10 min for
complete secondary and tertiary structure denaturation.
5. Before long-term storage at À80
C, purified RNA is quantified by spectrophotometry through measurement of the optical
density (OD) at 260 nm.
3.4.3 RT-qPCR on Total
RNA from Blood and
Organs
1. Approximately 1 μg of total extracted RNA from blood and
tissue from infected and control animals are retro-transcribed
into cDNA using the Transcriptor First Strand cDNA synthesis
kit. Program for cDNA synthesis is as follows: activation step,
25
C, 10 min; RT step, 55
C, 30 min; and inactivation of
enzyme, 85
C, 5 min. cDNA can be conserved between +4
C
and À20
C until qPCR assays.
Hamsters as a Model to Study Leptospirosis
251
equivalents per μg of DNA extracted from tissue samples or
blood (see Note 12).
3.4 Cytokine Gene
Expression Analysis
All steps should be carried out using a refrigerate block or on ice to
avoid RNA degradation, except otherwise cited.
3.4.1 Total RNA
Extraction from Blood
1. Total RNA is purified from blood collected as described in
Subheading 3.2 and stored in PAXgene collector tubes using
the PAXgene blood RNA system according to manufacturer’s
instructions.
2. Following the washing steps, RNA is eluted in 80 μL elution
buffer, and 40 μL of RNA eluate is treated for 30 min at 37
C
with TURBO DNase (2 units) for elimination of residual
genomic DNA.
3. Incubate the DNase-treated RNA at 65
C for 10 min for
complete secondary and tertiary structure denaturation.
4. Before long-term storage at À80
C, purified RNA is quantified by spectrophotometry through measurement of the optical
density (OD) at 260 nm.
3.4.2 Total RNA
Extraction from Organs
1. Prior to RNA extraction from organs collected as advised in
Subheading 3.2, tissue samples kept in RNAlater reagent are
first disrupted and homogenized using the MagNA Lyser
Instrument during 50 s at 4000 Â g and then collected after
centrifugation for 2 min at 18,000 Â g.
2. Total RNA is extracted from tissue lysates using the High Pure
tissue RNA Isolation Kit.
3. Following the washing steps, RNA is eluted in 80 μL elution
buffer, and 40 μL of RNA eluate is treated for 30 min at 37
C
with TURBO DNase (2 units) for elimination of residual
genomic DNA.
4. Incubate the DNase-treated RNA at 65
C for 10 min for
complete secondary and tertiary structure denaturation.
5. Before long-term storage at À80
C, purified RNA is quantified by spectrophotometry through measurement of the optical
density (OD) at 260 nm.
3.4.3 RT-qPCR on Total
RNA from Blood and
Organs
1. Approximately 1 μg of total extracted RNA from blood and
tissue from infected and control animals are retro-transcribed
into cDNA using the Transcriptor First Strand cDNA synthesis
kit. Program for cDNA synthesis is as follows: activation step,
25
C, 10 min; RT step, 55
C, 30 min; and inactivation of
enzyme, 85
C, 5 min. cDNA can be conserved between +4
C
and À20
C until qPCR assays.
Hamsters as a Model to Study Leptospirosis
251