7900ht real time pcr system

Adapter-ligated RNAs were incubated at 95°C for 5 min to denature, and then placed on ice. The denatured adaptor-ligated RNAs were reverse-transcribed using ReverTra Ace (TOYOBO) using a reverse transcription (RT) primer (Supplemental Table S1). The sequence of RT primer was based on the sequences of the adenylated linker and an additional sequence from nonhuman species (Gryllus bimaculatus) (Tsukamoto and Nagata 2016 (link)), since tRNA sequence as a template is too short to prepare a primer and probe set for qPCR. RT reaction was performed at 42°C for 60 min. The resultant cDNA solution was diluted with water by 1:5, and 1 µL of this solution was added to the real-time PCR mixture containing 5 µL of 2× TaqMan Genotyping Master Mix (Thermo Fisher Scientific), 0.4 µL of Custom TaqMan SNP Genotyping Assays containing specific primers and probes (Supplemental Table S3) (Thermo Fisher Scientific), and 3.6 µL of distilled water. 7900HT Real-Time PCR System (Thermo Fisher Scientific) was used to determine the threshold cycles (C_t). The cycling conditions were as follow: initial denaturation for 10 min at 95°C, 40 or 50 cycles of 5 sec at 95°C, and 60 sec at 60°C. Fluorescence signals were collected at the 60°C step of each cycle. All reactions were run in duplicate and the threshold cycles were determined.

Large RNA from the cells which were extracted using Isogen II (Nippon gene) or TRIzol (Thermo Fisher Scientific) according to the manufacturer's protocol was used as the template. Extracted RNA was treated with RQ1 DNase I (Promega) at 37°C for 30 min. The RNA quality and quantity were determined by a NanoDrop 2000 (Thermo Fisher Scientific). Then, RNA (300 ng each) was reverse-transcribed using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). The reverse transcription mixture was incubated at 42°C for 60 min. The resultant cDNA solution was diluted with water by 1:5, and 1 µL of this solution was added to the real-time PCR mixture containing 5 µL of 2× GeneAce SYBR qPCR Mix α (Nippon gene), 0.5 µL of each primer in 10 µM, and 3 µL of H₂O. 7900HT Real-Time PCR System (Thermo Fisher Scientific) was used. The cycling conditions were as follows: initial denaturation for 10 min at 95°C, 40 cycles of 15 sec at 95°C, 30 sec at 60°C. Fluorescence signals were collected at the 60°C step of each cycle. All reactions were run in duplicate and the C_t values were determined. The expression levels of target genes were normalized to the expression levels of ActB. To evaluate the reaction using the ΔC_t method, we used a primer set shown in Supplemental Table S4.