Applied biosystems 2720 thermal cycler

Total RNA was obtained using NZYol according to manufacturer’s protocol, with minor modifications. Bovine monocytes were lysed with 200 µL NZYol and incubated with 0.1 µg/mL RNA-grade Glycogen (Thermo Fisher Scientific). Each sample was incubated with 80 µL chloroform for phase separation and 200 µL isopropanol was added for RNA precipitation. The precipitated RNA was washed with 70% ethanol and resuspended in 5 µL RNase-free water. Synthesis of first-strand cDNA was done in an Applied Biosystems^® 2720 Thermal Cycler (Thermo Fisher Scientific) at 25°C for 10 min, 50°C for 30 min, and 85°C for 5 min using NZY First-Strand cDNA Synthesis Kit, according to manufacturer’s instructions (NZYtech). Samples were kept at -20°C. Negative controls using RNA samples for cDNA synthesis without reverse transcriptase (no RT control), and with no added template (no template control) were also included for all primer pairs.

cDNA synthesis and qPCR were performed using a previously reported method^{30 (link)}, with slight modifications. Briefly, cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio) with oligo(dT) primers and 1 μg of total RNA. The synthesized cDNA was mixed with SYBR Premix Ex Taq II (Takara Bio) and specific primers. Amplification was performed for 50 cycles, each at 95 °C for 15 s and 60 °C for 1 min, using a StepOnePlus real-time PCR system (Thermo Fisher Scientific). The list of primers used is presented in Supplementary Table S7. All measurements were performed in duplicate. For quantifying the levels of GAPDH and RPS18, a calibration curve was generated using 10, 5, 2.5, 1.25, 0.625, and 0.3125 fM synthetic DNA, which included the target sequence (GAPDH, GenScript Japan, Tokyo, Japan; RPS18, Integrated DNA Technologies, Coralville, IA, USA). The list of synthetic DNA sequences used is provided in Supplementary Table S8. For gel images, amplification was performed for 35 cycles, each at 95 °C for 15 s and 60 °C for 1 min, using an Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific).

Genomic DNA was purified from cultured thyroid cells (Nthy-ori 3-1, K1, TPC-1 and BCPAP) using QIAamp DNA mini kit (Catalog number, 51304) purchased from Qiagen (Hilden, Germany) and quantified with the Nanodrop spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). Next, we designed specific primers able to amplify the exonic region of genes containing the high impact mutations. PCR was carried out on Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific) using Taq DNA polymerase (Catalog Number EP0401, Thermo Fisher Scientific) with the following thermal cycles: denaturation at 95 °C (for 3 min), 40 cycles at 95 °C (for 30 s), 60 °C (for 30 s), 72 °C (for 1 min), followed by 72 °C (for 5 min).
The genes, the exons harboring the high impact mutations found in PTC patients, and the primer sequences used are listed in Additional file 1: Table S1.
The amplicons sequencing was performed as follows: 8 μl of primer forward (concentrated 2 μM) and 20 μl of DNA (concentrated 16 ng/μl). Sanger electropherograms were analyzed to evaluate the presence/absence of the indicated mutations.

Total genomic DNA were extracted using the DNeasy Power Soil Pro Kit (QIAGEN, Frankfurt, Germany) and stored at −20 °C prior to further analysis. A NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify DNA; the quality of DNA was detected by 1.2% agarose gel electrophoresis. For PCR amplification of the bacterial 16S rRNA genes at the V3–V4 region, the primers were 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [42 ]. For the ITS1 regions of fungi, the primers were ITS1F (5-GGAAGTAAAAGTCGTAACAAGG-3) and ITS2 (5-TCCTCCGCTTATTGATATGC-3) [43 (link)]. The PCR amplification was performed by Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA); the Quant-iT PicoGreen dsDNA assay was performed by a Microplate Reader FLx800 (BioTek, Burlington, VT, USA). PCR amplicons were purified and recovered by adding Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified with the fluorescent reagent of Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA, USA); then, amplicons were mixed in proportion to the sequencing amount, and pair-end 2 × 250 bp sequencing was performed using the Illlumina MiSeq platform with a NovaSeq 6000 SP reagent kit at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

Total RNA was isolated using the TRI Reagent^® (Molecular Research Center, Inc., Cincinnati, OH, USA; Cat. No. TR 118) according to the manufacturer’s protocol, and quality-checked with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Next, DNase I treatment was performed, and 1000 ng of total RNA was reverse-transcribed into complementary DNA (cDNA) by using the High-Capacity cDNA Reverse Transcription Kit (both obtained from Thermo Fisher Scientific; Cat. No. AM2222 and 4368813) on an Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific), according to the manufacturer’s protocols [88 (link),89 (link),90 (link),91 (link),92 (link),96 (link)].

The procedure stated by Rishniw et al. was used with some modifications using Promega reagents [19 (link)]. The pan-filarial primers (DIDR-F1 5′-AGT GCG AAT TGC AGA CGC ATT GAG-3′ and DIDR-R1 5′-AGC GGG TAA TCA CGA CTG AGT TGA-3′) that spanned the ITS2 of the rDNA designed by Rishniw et al. were employed to amplify the target DNA region. Known positive and negative controls were included in each PCR reaction. The PCR procedure consisted of an initial heating step at 94°C for 2 minutes, 32 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds and a final extension at 72°C for 7 minutes and a soak at 4°C in an Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific).
Discrimination of the six species was based on the size of the amplified PCR products. DIDR-F1 and DIDR-R1 primers amplified 484 bp, 542 bp, 578 bp, 584 bp, 615 bp, and 664 bp products from Dirofilaria repens, Dirofilaria immitis, Acanthocheilonema reconditum, Acanthocheilonema dracunculoides, B. malayi, and B. pahangi, respectively [19 (link)].