Abi prism 7500 fast

Each pool, containing the corresponding of 5 digestive tracts of dissected R. prolixus, were pretreated for 2 h at 56°C with 100 μl lysis buffer containing 10 mM Tris-HCl (pH 9.2), 1 mM EDTA and 150 μg/ml proteinase K (Sigma-Aldrich, St. Louis, MO, USA). DNA was purified from the lysate using QIAamp DNA mini kit (Qiagen, Hilden, Germany) and resuspended from the silica column in a final volume of 100 μL of elution buffer from the kit [11 (link)]. DNA was stored at– 20°C until further analysis.
Multiplex qPCR reactions were carried out in a final volume of 20 μl, containing 2 μl DNA (8–10 ng), 2× FastStart TaqMan Probe Master Mix (Roche applied science, Mannheim, Germany), 600 nM cruzi1/cruzi2 primers and 250 nM Cruzi3 probe (FAM/NFQ-MGB) targeting T. cruzi nuclear satellite DNA (SAT-DNA), 300 nM P2B primer, 500 nM P6R primer and 150 nM Triat Probe (VIC/NFQ-MGB) (Applied Biosystems) targeting the 12S ribosomal subunit gene of triatomines [11 (link),19 (link),22 (link)]. Sequences of both sets of primers and probes are presented in Table 1. The cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 58°C for 1 min. Amplifications were performed in the ABI Prism 7500 Fast (Applied Biosystems).

qRT-PCR was performed as previously described [32 (link)]. Briefly, 5 µg of total RNA was reverse transcribed using SuperScript III First-Strand Synthesis (Thermo Fisher, Waltham, MA, USA) following Total RNA extraction by Trizol (Invitrogen, Thermo Fisher, Waltham, MA, USA). Real-time PCR was performed on the ABI Prism 7500 Fast instrument (Applied Biosystems, Thermo Fisher, Waltham, MA, USA) using Sybr Green I Mastermix (Thermo Fisher, Waltham, MA, USA). Samples were tested in triplicates, three independent times and GAPDH was used as endogenous control product. Primer sequences were as follows: GAPDH forward 5′-GCATCTTCTTTTGCGTCGCC-3′ and reverse 5′-GCGCCCAATACGACCAAATC-3′, TRPC1 forward 5′-GAGCAGAGGATGACGTGAGG-3′ and reverse 5′-CCCAGGAAGAGGACGAGAGA-3′, TRPC4 forward 5′-AACAATAGGGAGGCGAGCTG-3′ and reverse 5′-CGGTCAGGOCCTTCTTCAGTT-3′, and TRPC5 forward 5′-GCCACACCTTGIAGGACCTC-3′ and reverse 5′-CTGCCCACGTACACTAAGCA-3′.

Total RNA was isolated according to the Tri Reagent acid: phenol protocol as specified by the supplier (Molecular Research Center Inc.). The reverse transcription (RT) and quantitative PCR reactions were performed as previously described [11 (link), 26 (link)]. The real-time PCR amplifications were performed using an ABI Prism 7500FAST instrument from Applied Biosystems (Foster City, CA). For each reaction, the final volume of 20 μL comprised 10 μL of SYBR Green PCR Mix, 2 μL (4 μM) of each previously reported primer [11 (link)], and 6 μL of the indicated dilution of RT products. For each gene in each tissue, the amplification efficiency was tested using 2 to 5 log of cDNA produced from liver- or kidney-purified RNA and sequential dilutions of UGT cDNA constructs (0.0001 to 10 pg/μl). The difference between standard curve and sample efficiency was below 10%, as recommended [27 (link)]. The amount of target genes was derived from linear regression with standard curves of these UGT constructs according to reported protocols [28 (link)]. Messenger RNA levels were calculated using the average molecular weight of one base-paired nucleotide (660 g/mol) and Avogadro's constant (6.022 × 10²³ mol⁻¹).

To confirm the results of the DGE analysis, the expression of 20 selected genes was measured using qPCR. cDNA was synthesized using the SYBR PrimeScript reverse transcription-PCR kit II (Takara). qPCRs were performed in 96-well plates using the ABI Prism 7500 fast real-time PCR system (Applied Biosystems) with SYBR green detection. Each gene was analyzed in triplicate, after which the average threshold cycle (C_T) was calculated per sample. The relative expression levels were calculated using the 2^−ΔΔCt =  2^{−[ΔCt (treatment)− ΔCt (control)]} method. The reference gene actin was used to normalize the expression level of other genes [37] (link). All of the designed primers were synthesized at Boshang BioCompany (Table S1).

Total RNA from cell lines or primary islets was isolated after the indicated treatments by using the High Pure RNA Isolation Kit (# 11828665001, Roche Diagnostics, Risch-Rotkreuz, Switzerland) and cDNA was prepared from 500 ng RNA (for cells) or 150 ng RNA (for islets) using oligo-d(T) and superscript II reverse transcriptase (# 18064014, Invitrogen). qRT-PCR was performed in a total volume of 10 µl, containing 4 pmol of forward and reverse primers, 5 µl Fast SYBR® Green Master Mix (# 4385612, Applied Biosystems, CA, USA), and 0.2 μl cDNA. The samples were assayed on an ABI-prism 7500 Fast (Applied Biosystems). The relative expression level of the genes of interest was normalized to the geometrical mean of three housekeeping genes, namely, hypoxanthine-guanine phosphoribosyltransferase (Hprt), 60S ribosomal protein L27 (RPL27), and beta-actin. The ∆∆Ct method was used for quantification. The amplification efficiency was equal for all genes. The primer sequences are provided in Supplementary Table S2.