Nancy 520

Total RNA was extracted from cardiac tissue using standard guanidinium thiocyanate-phenol-chloroform RNA extraction methods.
RNA samples were treated with RQ1 RNase-free DNase (Promega), according to the manufacturer’s instructions. Samples were then reverse transcribed to cDNA using M-MLV Reverse Transcriptase (Promega) with Random Primers (Promega), according to the manufacturer’s instructions. ‘No reverse transcriptase’ controls were created for each RNA sample by using water in place of the reverse transcriptase enzyme.
PCR was carried out according to standard protocols using GoTaq polymerase (Promega) in combination with dNTPs and specifically designed primers. Primers were designed using PrimerSelect (Lasergene 8; DNASTAR Inc.). The primer sequences used to detect TMSB4X transcripts were as follows.
Forward primer: GAAGACAGAGACGCAAGAGAAAAAReverse primer: TGCCAGCCAGATAGATAGACAGAT.
PCR was carried out using a G-Storm GS1 thermocycler, using an annealing temperature of 58°C. The PCR products were resolved on 2% agarose gels containing Nancy-520 (Sigma Aldrich). Gels were imaged using a High Performance Ultraviolet Transilluminator (UVP) and the associated DocIT software (UVP).
DOI: dx.doi.org/10.17504/protocols.io.rvbd62n

Snail ITS2 fragment was amplified using snail-specific primers following the protocol established by Bargues et al. [23 ]. Two sets of primers were employed to amplify the 28S rDNA and COX1 regions, as specified by Sandoval et al. [25 ] and Lockyer et al. [26 ], respectively, for analyses of emerging cercariae.
PCR was conducted using a 50 μL reaction volume and GoTaq Flexi DNA Polymerase (Promega, UK) consisting of 200 µM dNTP mix, 1.5 mM MgCl₂, 10 pmol of each primer, 1.25 U of Taq DNA polymerase, and 1 µL of extracted DNA template. Amplification was performed using a GeneAmp PCR System 9700 (Applied Biosystems, USA). The primer sequences and PCR conditions used in this study are described in Table-1 [23 , 25 , 26 ].
Amplified PCR products were analyzed using 1.5% agarose gel electrophoresis in tris, acetate, and EDTA. The agarose gel was pre-stained with Nancy-520 (Sigma-Aldrich, UK). PCR fragments of the positive samples were randomly selected for sequencing. DNA was sequenced in both directions using forward and reverse primers. Sequencing was performed using the U2Bio sequencing service (Korea).

Total DNA was extracted with CTAB buffer with added 2% (w/v) polyvinylpolypyrrolidone. The ITS region of the rDNA was amplified by PCR using the general primers53 (link), gITS7 and ITS4. Each sample was uniquely barcoded. PCR was performed in 50 μl reactions and consisted of the following final concentrations, 0.25 ng μL⁻¹ template DNA, 200 μM of dNTPs; 750 μM of MgCl₂; 0.025 μM polymerase (5 U/μL) (DreamTaq Green, Thermo Scientific, Waltham, USA), and 200 nM of each primer in 1× buffer. Amplifications were performed using the Applied Biosystems 2720 thermal cycler. The PCR program started with denaturation at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, annealing at 56 °C for 30 s and 72 °C for 30 s, followed by a final extension step at 72 °C for 7 min. To check if the PCR was successful, the PCR products were visualized by gel electrophoresis on 1% agarose gel stained with Nancy-520 (Sigma-Aldrich, Sweden). PCR products were purified using Agencourt AMPure XP (Agencourt Bioscience Corp, Massachusetts USA) and with the ‘E.Z.N.A. Cycle-Pure’ kit (Omega) following manufacturer’s instructions. After quantification of PCR products using a Qubit flurometer 2.0 (Life Technologies, Sweden), samples were pooled in an equimolar mix and sent for 454-amplicon sequencing using the GS FLX Titanium chemistry (Macrogen Inc, Seoul, Korea).

Penicillin, streptomycin, Schneider's Drosophila medium, cell culture flasks, and the fluorescent stain Nancy-520 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Fetal calf serum (FCS), primers for proteases, kDNA3 and housekeeping genes, TRIzol reagent, RNase H enzyme, DEPC-treated water, deoxyribonucleotide phosphate solution (dNTPs), Platinum Taq DNA Polymerase (DNA Polymerase), and Taq Platinum PCR buffer were purchased from Invitrogen, Life Technologies (USA). iScript cDNA Synthesis kit was purchased from Bio-Rad Laboratories, (Hercules, CA). GoTaq® qPCR Master Mix, Wizard SV Gel Kit, and PCR Clean-UpSystem were purchased from Promega Corporation (USA). High Pure PCR Template Preparation Kit was purchased from Roche Molecular Systems, Inc. Chloroform and ethanol were purchased from Merck (Brazil). All reagents were of analytical grade or higher.

Total RNA was isolated using TRIzol Reagent (Invitrogen) from whole heart tissue and cell pellets of cultured primary ventricular fibroblasts. 1 μg of RNA was used for cDNA synthesis using M-MLV reverse transcriptase (Promega). RT-PCR was carried out using Go-Taq polymerase (Promega) in a 50 μl reaction containing 0.5 μM primers, 10 mM dNTPs and 5 μl of 10 ng/μl cDNA. Primer sequences and amplicon sizes are listed in S2 Table. Polymerase Chain Reaction (PCR) consisted of initial denaturation of DNA at 95°C, followed by 40 cycles of 94°C for 1 minute, annealing at 58°C for 1 minute and extension at 72°C for 1 minute, with a final extension of 72°C for 10 minutes. The PCR products were resolved on 1.5% agarose gels containing Nancy-520 (Sigma Aldrich) and imaged using a high performance ultraviolet transilluminator (UVP) and the associated DocIT software.