Phenol chloroform isoamylic alcohol

Total DNA was extracted and purified from the complete body of adult ticks of each tick species after dissection into quarters. Before dissection, each sample was washed in different decreasing concentrations of alcohol (70%, 50%, 30%, 10%) for 1 h per each concentration and finally in double distilled water for 1 h. DNA isolation procedures were performed using high salt concentration protocol [61 ]. 20 mg from each tick tissues were incubated with lysis buffer (10 mM Tris-HCl, 10 mM EDTA, 3.4 mM SDS and 20 mM NaCl pH 8.0, Sigma-Aldrich), in addition to 1 M DTT (Sigma-Aldrich) and proteinase K (20 mg/ml) (Stratagin) at 56°C overnight, then the supernatant was transferred to new collecting tube (Coaster) to proceed in DNA isolation. Purity and concentration of isolated genomic DNA was measured by nanodrop 2000c (Thermo Scientific). Samples recording <1.5 ratio at 260/280 wavelength were proceeded to second phase of DNA isolation by shaking in phenol/chloroform/isoamylic alcohol (25:24:1, Sigma-Aldrich) and precipitated by adding 2 volumes of ice-cold ethanol for 4-6 h [62 ]. After centrifugation (at 14,000 rpm for 5 min), the pellet was resuspended in 50 µl Tris-EDTA buffer (10 mM Tris, pH 8.0 and 1 mM EDTA), and the DNA was used as template for PCR and the remaining DNA was stored at −20°C.

Total RNAs were extracted with Trizol from cell pellets containing 20 × 10⁶ cells. The aqueous phase was further extracted with phenol-chloroform-isoamylic alcohol (25:24:1; Sigma), then with chloroform. Total RNAs were recovered after precipitation with 2-propanol. For Northern blot analyses, RNAs were dissolved in formamide, denatured for 10 min at 70°C and separated on a 1.2% agarose gel containing 1.2% formaldehyde and 1× Tri/Tri buffer (30 mM triethanolamine, 30 mM tricine, pH 7.9) (3 μg RNAs/lane). RNAs were transferred to a Hybond N⁺ nylon membrane (GE Healthcare) by passive transfer. Pre-hybridization was performed for 1 h at 45°C in 6× SSC, 5× Denhardt's solution, 0.5% SDS, 0.9 g/ml tRNA. The 5′-radiolabeled oligonucleotide probe was incubated overnight. The sequences of the probes were: 5′ITS1 (5′-CCTCGCCCTCCGGGCTCCGTTAATGATC-3′), ITS1-5.8S (5′-CTAAGAGTCGTACGAGGTCG-3′), ITS2 (ITS2b: 5′-CTGCGAGGGAACCCCCAGCCGCGCA-3′ and ITS2d/e: 5′-GCGCGACGGCGGACGACACCGCGGCGTC-3′), 18S (5′-TTTACTTCCTCTAGATAGTCAAGTTCGACC-3′), 28S (5′-CCCGTTCCCTTGGCTGTGGTTTCGCTAGATA-3′). Membranes were washed twice for 10 min in 2× SSC, 0.1% SDS and once in 1× SSC, 0.1% SDS, and then exposed. Signals were acquired with a Typhoon Trio PhosphoImager (GE Healthcare) and quantified using the MultiGauge software.

1x10⁷ adhered cells infected or not with promastigotes, at different periods of infection, were collected using cell scrapers (Sarstedt) alongside eventually detached cells present in culture medium. Cells were washed twice with ice-cold PBS, lysed with lysis buffer (40 mM EDTA pH8, 50 mM Tris pH 8, 1% Triton) and incubated with RNAse (20μg/mL) (Promega) for 1h at 37°C. Proteinase K (Promega) was added to a final concentration of 100μg/mL and incubated at 56°C for at least 2h. Supernatants were treated twice with phenol: chloroform: isoamylic alcohol (25:24:1) (Sigma-Aldrich), centrifuged at 15000 rpm for 5 minutes at 4°C and finally treated with chloroform (Sigma-Aldrich) before overnight incubation at -20°C with 0.25M NaCl and twice the volume of pure ethanol (Promega). Samples were washed twice with ethanol 70% and the dried pellet was solubilized in TE (10 mM Tris-HCl, pH 8.0; 10 mM EDTA, pH 8.0). DNA samples were quantified using GeneQuant (Amersham, GE Healthcare Biosciences) and 1 μg of DNA of each sample was applied in 1.8% agarose gels. Gels were analyzed and documented with UV transilluminator (Stratagene-Agilent Technologies) and a camera (Vilmer Loumart).