Hot firepol evagreen qpcr mix plus

Total mRNA was extracted from 30 larvae using Trizol (Invitrogen) and reverse-transcribed using SuperScript® III First-Strand (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed in triplicated using a Rotor-gene Q (Qiagen) and 5× HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne), following the manufacturers’ protocols. The cycling parameters were 95 °C for 14 min, followed by 45 cycles at 95 °C for 15 s, 59 °C for 20 s and 72 °C for 20 s. Threshold cycles (Ct) and dissociation curves were generated automatically by Rotor-gene Q series software. Sample Ct values were normalized with Ct values from zebrafish actb2 and rplp0, used as control genes. Primer sequences are listed in Supplementary Table 1. All primers were designed using the software Primer3^{19 (link)}.

10 mg rat liver or soleus muscle was extracted for total RNA using an RNA extraction kit (FavorPrep^TM Blood/Cultured Cell Total RNA Purification Mini Kit, Favorgen Biotech, Ping-Tung, Taiwan) and transcribed to cDNA using the FIREScript RT cDNA Synthesis KIT (Solis Bio-Dyne, Tartu, Estonia) with oligo (dT) 18 primer following manufacturer’s instructions. For real-time PCR, samples of cDNA were triplicate analyzed using the 5× HOT FIREPol^® EvaGreen^® qPCR Mix Plus (Solis Bio-Dyne, Tartu, Estonia) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The levels of relative mRNA were analyzed and normalized to β-actin (Actb), used as an endogenous control gene. The final concentration of primers in the reaction was 250 nM, and their sequences are presented in Table 1. Thermal cycling for PCR was as follows: activation of 94 °C for 15 min, followed by 40 cycles of amplification at 94 °C for 15 s, 54–59 °C for 20 s, and 72 °C for 27 s.

The genome sequences of T4, A3R, and 676Z phages were obtained from the GenBank (accession numbers for T4, A3R, and 676Z are NC_000866, JX080301, and JX080302, respectively). Based on the genome sequence, qPCR primers were designed using the Primer-BLAST software at the National Center for Biotechnology Information (National Center for Biotechnology Information, 2017 ).
To detect T4 phage we used forward primer 5′-ACT GGC CAG GTA TTC GCA-3′ and reverse primer 5′-ATG CTT CTT TAG CAC CGG CA-3′. To detect A3R and 676Z phages the following primers were used: forward primer 5′-TGA AGA AGA CCG TGC AGG ATT-3′ and reverse primer 5′-TCA GAA GGA GCT GAT TGA GCG-3′.
The amount of genomic DNA in each test sample was determined using 5× HOT FIREPolEvaGreen qPCR Mix Plus (Solis BioDyne, Tartu, Estonia) following the manufacturer’s recommendations. Briefly, each PCR reaction contained 1 μl of DNA template and 15 pM of each primer as well as 2 μl of 5× HOT FIREPolEvaGreen qPCR Mix Plus in a final volume of 10 μl. Cycling conditions were as described by 5× HOT FIREPolEvaGreen qPCR Mix Plus’s manufacturer. The amount of phage genomic DNA in test samples was calculated based on the standard curve generated using standard solutions prepared as outlined above. qPCR normalization was performed according to MIQE Guidelines (Bustin et al., 2009 (link)).