Viia7 thermocycler

P. ovale spp. isolates were selected from the FNMRC database based on parasite densities and the countries where the patients got contaminated.
Genomic DNA was extracted from 200 μL of whole blood using MagNA Pure automaton (Roche diagnostics, USA) and eluted in 100 μL. P. ovale spp. mono-infection was confirmed with the species-specific quantitative PCR (qPCR) Plasmodium typage kit (Bio-Evolution, France) targeting the 18s rRNA for P. ovale spp. and the human beta actin gene to evaluate human DNA contamination. The reaction was carried out on a ViiA 7 thermocycler (Applied Biosystems). One positive and one negative control were included in each run. ΔCt (cycle threshold) was defined as the difference between the Ct of P. ovale spp. and the human Ct. We performed in-house qPCR high resolution melting (HRM) to differentiate P. ovale wallikeri from P. ovale curtisi (42 (link)).

The discovery cohort consisting of four DAWT, eight FHWT, and six non-neoplastic control patient autopsy samples were included. All samples used in this phase were frozen tissue. cDNA was synthesized from 1 μg of total RNA using a Megaplex kit (4444745, Applied Biosystems). To create a miRNA expression profile, 756 miRNAs were divided into cardA v2.0 and B v3.0 and tested following the manufacturer's instructions. TaqMan MicroRNA Master Mix was used for amplification, performed in a Viia 7 thermocycler (Applied Biosystems). MiRNAs with Cq values >35 were considered non-informative and were excluded.
Tumoral Cq values were normalized to those of U6 snRNA and RNU48 endogenous controls and compared with those of non-neoplastic kidney tissues using the relative quantification formula 2^−ΔΔCt. Heatmaps for the relative expression values (fold change) were generated using MultiExperiment Viewer (MeV) v.4.6. Data were evaluated by comparing expression values between DAWT, FHWT, and control samples. The SAM (Significance Analysis of Microarrays) program was used to evaluate significant differences between miRNAs groups. Values >2 were considered to correspond to upregulation, values < −2 to downregulation, and values between −2 and 2 to no change in expression.

The diseased P. vannamei examined in this study were collected from 2016 to 2019 from affected ponds located in the Brazilian northeast region including Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State. The average weights of the diseased juvenile shrimp in the affected ponds were approximately 3.0 gm. Five to six shrimp were collected from each farm and screened by a real-time RT-PCR analysis, H&E histopathology and ISH. RNA isolation was performed using approximately 25 mg of the minced muscle and pleopods of the presumably affected shrimp. The Tissue LEV Total RNA Purification Kit (#AS1220, Promega, Madison, WI, USA) along with an automated DNA/RNA extraction system (Maxwell^® MDX Promega, USA) was used following the manufacturer’s recommendations. One-step real-time RT-PCR was carried out following the protocols described by Andrade et al. [16 (link)]. Prior to the real-time assay, extracted RNA was boiled at 100 °C for 3 min to denature dsRNA, then placed on ice and finally subjected to real-time RT-PCR analysis using GoTaq^® Probe 1-Step RT-qPCR system (PROMEGA, Madison, WI, USA). Each sample was analyzed in duplicate using a real-time PCR ViiA⁷ thermocycler (Applied Biosystems, Foster city, CA, USA).

Bronchoalveolar lavage and measurement of the total protein and cytokines in BALF were performed as previously described in [26 (link),28 (link)]. For the histology, after bronchoalveolar lavage (BAL), the lungs were excised and the left lobe fixed in 4% buffered formalin and embedded in paraffin. Sections of 3 µm thickness were stained with hematoxylin-eosin (H&E) and periodic acid Schiff (PAS). For the real-time PCR, total RNA was extracted from snap-frozen lung tissue as in [25 (link)]. A total of 0.4 μg of RNA was converted to cDNA following manufacturing protocol (Fermentas, ThermoFischer Scientific, Waltham, MA, USA). Real-time PCR was performed with SYBR Green and ViiA™ 7 thermocycler (Applied Biosystems, Foster City, CA, USA). Relative expression levels were calculated using the 2^ddct method, normalized to ACTB. The primer sequences were the following: Arg1 (forward) 5′-AGAGATTATCGGAGCGCCTT-3′ (reverse) 5′-TTTTTCCAGCAGACCAGCTT-3′; Muc5ac (forward) 5′-TGGAGTCAGCACGAAAACAG-3′ (reverse) 5′-GCACTGGGAAGTCAGTGTCA-3′; IL-13 (forward) 5′-TGTGTCTCTCCCTCTGACCC-3′ (reverse) 5′-CACACTCCATACCATGCTGC-3′; KC (forward) 5′-CCACACTCAAGAATGGTCGC-3′ (reverse) 5′-TCTCCGTTACTTGGGGACAC-3′; ACTB (forward) 5′-TTCTTTGCAGCTCCTTCGTT-3′ (reverse) 5′-ATGGAGGGGAATACAGCCC-3′. Data were considered significant with a p value ≤ 0.05 in a confidence interval of 95%.