Kapa sybr fast qpcr kit

To validate the target genes of interest, i.e., those predicted by both the microarray and in silico analyses, qRT-PCR was conducted using transcript-specific primers designed as shown in Table S2. Total RNA was extracted from the cells immediately (day 0), 3, 6 and 10 days after RA treatment. Then, 500 ng of total RNA was transcribed into cDNA using the miScript II RT Kit (Qiagen) and was detected by the KAPA SYBR Fast qPCR Kit (Nippon Genetics, Tokyo, Japan). The relative mRNA expression level was determined by qRT-PCR, and the expression levels were normalized to the average GAPDH expression level using the ΔΔCT method.

Female medaka of esr2a^+/− and esr2a^−/− (age: three to four months) were deeply anesthetized and their pituitaries were collected for qRT-PCR.
Total RNA was extracted from the pituitaries using the NucleoSpin RNA plus (TaKaRa, Kusatsu, Japan) according to the manufacture’s instruction. Total RNA samples were then reverse transcribed by PrimeScript^TM RT Master Mix (TaKaRa). For qRT-PCR, the cDNA was amplified by KAPA SYBR FAST qPCR kit (Nippon Genetics, Tokyo, Japan) with AriaMX Realtime PCR System (Agilent Technologies, Santa Clara, CA). The temperature profile of the reaction was 95 °C for 3 minutes, 40 cycles of denaturation at 95 °C for 10 seconds, annealing at 60 °C for 10 seconds, and extension at 72 °C for 10 seconds. The PCR products were verified using melting curve analysis. The data were normalized by a housekeeping gene, ribosomal protein s13 (rps13). The melting curve analyses were conducted to ensure that the amplicons were the same as the sequence-certified ones. The primer pairs used in the real-time PCR are listed in the primer list (Supplementary Table 1).

Total RNA was extracted using a NucleoSpin® RNA Plus kit (Takara Bio Inc., Shiga, Japan). Reverse transcription was performed with a PrimeScript II 1st strand cDNA Synthesis Kit (Takara). qPCR was performed with a KAPA SYBR Fast qPCR kit (Nippon Genetics, Tokyo, Japan). All reactions were carried out on a LightCycler®96 (Roche). Each sample was analyzed in triplicate at least twice for each PCR measurement. Melting curves were checked to ensure specificity. Relative quantification of mRNA expression was calculated using the standard curve method with the GAPDH level. Before qPCR, the DNA fragment amplified using each primer set was detected to be a single band with the correct size by agarose gel electrophoresis. The following primer sets were used to amplify cDNAs; 5′-CAGCCCACCCTCAATACCAG-3′ and 5′-AGAAGGTGTTTGTGGCTGCT-3′ for mouse VEGF-C [15 (link)], 5′-TGCAGTGGCAAAGTGGAGATT-3′ and 5′-TGCCGTTGAATTTGCCGT-3′ for mouse GAPDH [16 (link)].

Following the standard protocol, total RNAs were extracted from the gill and adipose fin tissues of the fish samples in the HT and NT groups using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). RNA quality and integrity were assessed on an Agilent 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) using RNA ScreenTape. RNA concentrations were measured by a Qubit^® 2.0 Fluorometer RNA assay kit (Life Technologies, Carlsbad, CA, USA). Approximately 2 µg of total RNA from each sample was used to construct complementary DNAs (cDNAs) with SuperScript II (Invitrogen, Carlsbad, CA, USA). We prepared cDNA libraries using a TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Index adapters were added to identify sequences for each sample in the final data. The quality of the libraries was measured on an Agilent 2200 Tapestation using High Sensitivity RNA ScreenTape, and qPCR was measured using a KAPA SYBR Fast qPCR Kit (NIPPON Genetics, Tokyo, Japan). Subsequently, the 40 libraries (10 biological replicates per group (HT and NT), per tissue (gill and fin)) were subjected to paired-end (2 × 100 bp) sequencing on the Illumina HiSeq 4000 Sequencing System (Illumina) at BGI, Japan, and all samples were sequenced in the same lane.

Real-time PCR was conducted using the KAPA SYBR Fast qPCR Kit (Nippon Genetics) with synthesized cDNA as the template. The reaction was performed on the CFX96 Touch Deep Well real-time PCR analysis system (Bio-Rad) using the following cycling conditions: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 3 s, and annealing/extension at 60 °C for 20 s. Each gene expression level was normalized to that of Gapdh and analyzed using relative cycle threshold (CT). The values are presented as mean ± standard deviation.