Viiatm7 real time pcr machine

Target gene for the assay was the V3-V4 hypervariable region of the 16S rRNA bacterial gene, defined by a 174 bp fragment using the following primers 341f 5 ′ -CCTACGGGAGGCAGCAG-3 ′ and 515r 5 ′ -ATTCCGCGGCTGGCA-3 ′ as described in López-Gutiérrez et al. (2004) (link). The Atlantic salmon elongation factor gene EL1-a (ELF) described in Bland et al. (2012) (link) was used as the reference gene in this assay, amplifying a 66 bp fragment using the primer set S-ELF.f 5'-GGCCAGATCTCCCAGGGCTAT-3' and S-ELF.r 5'-TGAACTTGCAGGCGATGTGA-3'.
Extracted DNA was diluted to a working concentration of 10 ng.µl for all samples. Real time PCRs were carried out in a ViiA TM 7 Real-Time PCR Machine (Applied Biosystems). qPCR was performed in a single-plex 25ul reaction containing 1.25 µl of 10 uM forward and reverse primer, 8 µl of RNase-free H 2 O, and 12.5 µl of SYBR Green qPCR Mastermix including hotstart Taq polymerase (Bioline). Each reaction contained 2 µl of normalized template DNA (30 ng.ul -1 ). PCR reactions were subjected to the following thermal cycling: 95

Total RNA was extracted from independent pools of micro-dissected zebrafish livers using the RNeasy Micro Kit (QIAGEN, #74004). RNA integrity was assessed by a High Sensitivity RNA ScreenTape assay (Agilent, #5067-5579) on a 2200 TapeStation. cDNA was generated from 1-10 µg RNA using the Superscript III First Strand Synthesis System (Invitrogen, #18080051) and oligo(dT) priming according to the manufacturer's instructions. RT-quantitative PCR (RT-qPCR) was performed using a SensiMix SYBR kit (Bioline, #QT605-05) on an Applied Biosystems ViiATM7 Real-Time PCR machine. Expression data were normalized by reference to hrpt1, b2m and tbp. LinRegPCR V11.0 was used for baseline correction, PCR efficiency calculation and transcript quantification analysis 61 (link) . Relative expression levels were calculated by the 2 -∆∆Ct method and all results were expressed as the mean ± SEM of three independent pools of biological replicates. Primer sequences are listed in Supplementary Table 2.