T4 gene 32 protein

Template RNA was diluted in 1 μL of lysis buffer, denatured for 1.5 min at 70 °C, and stored on ice. The reaction buffer for RT was modified using the PrimeScript RT reagent Kit (TaKaRa). A mixture containing 2 μL of conventional RT mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18 (Thermo Fisher), 8 pmol random hexamers (TaKaRa), and 1.5× PrimeScript enzyme mix in RNase-free water) or 2 μL of RT-RamDA mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18, 8 pmol random hexamers or NSRs, 0.2 U of DNase I Amplification Grade (Thermo Fisher), 100 ng of T4 gene 32 protein (Roche), and 1.5× PrimeScript enzyme mix in RNase-free water) was added to 1 μL of diluted, denatured template RNA. The mixture was agitated for 30 s at 2000 rpm using MixMate (Eppendorf) and incubated in a C1000 thermal cycler (Bio-Rad) at 25 °C for 10 min, 30 °C for 10 min, 37 °C for 30 min, 50 °C for 5 min, and 85 °C for 5 min. The RT product was diluted 1:9 in nuclease-free water and used for qPCR analysis.

Telomerase assays were performed by the standard Telomeric Repeat Amplification Protocol (TRAP) [62 (link)]. Briefly, lysates were prepared by powdering samples (Table 1) frozen in liquid nitrogen, followed by homogenization in 200 μl of ice-cold lysis buffer and incubation for 30 min on ice. Assay tubes were prepared by sequestering 0.1 μg of CX primer (5′-CCCTTACCCTTACCCTTACCTAA-3′) (Nihon Gene Research Laboratories Inc., Japan). The extracts, equivalent to 6 μg protein, were assayed in 50 μl of reaction mixture containing TS primer (5′-AATCCGTCGAGCAGAGTT-3′) (Nihon Gene Research Laboratories Inc., Japan), 1 μg of T4 gene 32 protein (Roche, Switzerland), and 2 units of Taq DNA polymerase (Takara Bio Inc., Japan). After 30 min of incubation at room temperature for telomerase-mediated extension of the TS primer, the reaction mixture was heated at 90°C for 90 s and then subjected to 31 polymerase chain reaction (PCR) cycles. The PCR product was then electrophoresed, and the gels were stained with SYBR Green I (Lonza, Switzerland) for 30 min.
We used ImageJ software version 1.39 to quantify the band densities. Relative values of telomerase activity in medaka samples were obtained by calibration with reference values from a human cancer cell line (SiHa cells, positive control). The value of the positive control was defined as ‘one’ [38 (link)].

Hypervariable rrs gene V5-V6 regions of about 280 bp were amplified with 784F 5′-AGGATTAGATACCCTGGTA-3′ and 1061R 5′-CRRCACGAGCTGACGAC-3′ primers (Andersson et al., 2008 (link)). These primers were selected in silico with the RDP Probe Match tool according to the following criteria (Cole et al., 2009 (link)): hybridization with 94% of sequences belonging to the RDP Bacteria domain database with good quality and ≥1200 bp long with 2 mismatches allowed in primer sequences. In addition, Basic Local Alignment Search Tool (BLAST) was used to check that the chosen primers did not match Ae. albopictus 18S and mitochondrial 16S rDNA gene sequences. PCR amplification was performed using 1.75 U of Expand High Fidelity Enzyme Mix (Roche, Switzerland) with 1× Expand High Fidelity Buffer (Roche, Switzerland), 0.06 mg mL⁻¹ of T4 gene 32 protein (Roche, France), 0.06 mg mL⁻¹ of bovine serum albumin (New England Biolabs, France), 40 μM of dNTP mix, 200 nM of each primer (Invitrogen, France) and 30 ng of template DNA. PCR products were purified with QiaAmp purification kit (Qiagen, France) following the manufacturer's recommendations.