Step one plus real time pcr apparatus

The leptospiral burden in urine was determined by quantitative real-time DNA PCR (qPCR). Total DNA was extracted from a drop of urine (5 to 100 µl) using the Maxwell 16 instrument and Cell LEV DNA purification kit (Promega). The qPCR reaction was calibrated using a known number of heat-killed L. interrogans. The DNA concentration was adjusted to around 100 ng in the qPCR reaction. Primers were designed in the peculiar lpxA gene of L. interrogans Fiocruz strain [17] (link) to specifically detect pathogenic Leptospira spp but not other spirochetes or bacteria, using Primer Express 3 software (Forward (Fw): 5′-TTTTGCGTTTATTTCGGGACTT-3′; Reverse primer (Rv): 5′-CAACCATTGAGTAATCTCCGACAA-3′; Probe: 5′-TGCTGTACATCAGTTTTG -3′). qPCR reactions were run on a Step one Plus real-time PCR apparatus using the absolute quantification program (Applied Biosystems), with the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 15 s and annealing temperature 60°C for 1 min, according to the manufacturer's instructions. Results were expressed as the number of Leptospira in 100 µl of urine. Observation and subcultures of Leptospira from fresh urines showed that shed bacteria were mobile and alive (data not shown).

Total L. monocytogenes RNA was extracted from mid-exponential cultures (OD₆₀₀≈ 0.2–0.3 for BHI media) or intracellular bacteria (t = 4 h) using RNease mini kit (Qiagen). RNA samples were reverse transcribed using ImProm-II^TM reverse transcription system (Promega) and specific cDNAs quantified by real-time PCR (RT-QPCR) as previously described [5 (link)] using Step One Plus real-time PCR apparatus and Step One V2.3 software (Applied Biosystems). The PCR signal was monitored using TaqMan probes for the PrfA-regulated genes hpt and actA, and Power SYBR Green master mix (Applied Biosystems) and gene-specific primers for fosX. Transcription values of the target genes were normalized using the housekeeping genes rpoB and ldh. Fold-changes in fosX expression were determined by the 2^–ΔΔCT method. The oligonucleotides used are shown in S4 Table.

RNA was isolated manually using TRI Reagent® (MRC, OH, USA). cDNA was prepared from total RNA (1 µg) using Quantabio, and a qScript^TM cDNA synthesis kit and PerfeCTa SYBR Green SuperMix were used for qPCR according to the manufacturer’s instructions (Quantabio, MA, USA). To prepare DNA-free RNA samples, RNA was treated with PerfeCTa® DNase I according to the manufacturer’s instructions (Quantabio, MA, USA). A StepOne Plus Real-Time PCR apparatus with StepOne Software v2.3 was used for analysis (Applied Biosystems, MA USA). Each experiment was performed in multiple technical duplications (n > 4) with in 4 biological repeats. Relative mRNA expression was analyzed according to the 2^(−ΔΔCt) calculation method.

The leptospiral burden in blood and urine was determined by quantitative real-time PCR (qPCR), as described [40 (link)]. The Maxwell 16 automat was used to extract total DNA from 50 μl of blood and from a drop of urine, using the Maxwell blood DNA and cell LEV DNA purification kits (Promega), respectively. Primers and probe designed in the lpxA gene of L. interrogans strain Fiocruz L1-130 [4 (link)] were used to specifically detect pathogenic Leptospira spp. [40 (link)]. qPCR reactions were run on a Step one Plus real-time PCR apparatus using the absolute quantification program (Applied Biosystems), with the following conditions for FAM TAMRA probes: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 15 s and annealing temperature 60°C for 1 min, according to the manufacturer’s instructions.

The extraction of total RNA from the tissues was performed using an RNeasy Mini Kit (Qiagen, Carlsbad, CA, USA), and then reverse transcription was conducted with a TIANScript Reverse Transcription Kit (TIANGEN Inc. Beijing, China). Quantitative PCR analyses in real time were carried out using a StepOnePlus Real-Time PCR apparatus (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s directions. The PCR procedure was performed as follows: incubation (95°C, 10 min), 40 cycles of denaturation (95°C, 15 s), and annealing and extension (60°C, 1 min). Quantification of amplicons was accomplished with SYBR Green fluorescence. The oligonucleotide primer pairs included ZO-1, claudin 3, occludin, Muc2, cryptdin, regenerating islet-derived protein (Reg3γ), interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), transforming growth factor beta (TGF-β), IL-6, and IL-10, which were synthesized by GENEWIZ, Inc. (Beijing, China). The sequences are summarized in Supplementary Table S1. Relative messenger RNA (mRNA) quantification was calculated using the 2^−ΔΔCT method.

RNA were extracted using Trizol (Invitrogen, Cergy-Pontoise, France). For monolayer cultures, Trizol was directly added to cell layer in the culture dish. For 3D culture, ten alginate beads were dissolved in 55 mM sodium citrate, 150 mM NaCl and gently centrifuged, before adding Trizol on the cell pellet. Next, extraction was performed according to the manufacturer’s conditions (Invitrogen, Cergy-Pontoise, France). Thereafter, RNA (1 μg) was treated with DNAse-I (Invitrogen, Cergy-Pontoise, France), and reverse transcribed into cDNA in the presence of oligodT and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reaction was carried out at 37 °C for 1 h followed by a further 10-min step at 95 °C. Amplification of the generated cDNA was performed by real-time PCR in Step One Plus Real Time PCR apparatus (Applied Biosystems) with appropriate primers. The relative mRNA level was calculated with the 2^–ΔΔCT method.