Nucleospin rna column kit

Total RNA was extracted from all cardiomyocytes lines with a Nucleospin RNA column kit (TakaraBio). Library preparation was performed as previously described.
¹¹ (link) Sequencing was performed on an Illumina HiSeq 2500 platform in 75‐base single‐end mode; Illumina CASAVA 1.8.2 software was used for base calling. Sequenced reads were mapped to the human reference genome sequence (hg 19) using TopHat ver. 2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 0.1.19. Fragments per kilobase of exon per million mapped fragments were calculated using Cuffnorm ver. 2.2.1. Ingenuity pathway analysis was used to identify canonical pathways from differentially expressed genes. Data from this study can be accessed under Gene Expression Omnibus experiment accession number (GSE240775). Transcriptomes were analyzed using the integrated differential expression and pathway platform analysis (http://bioinformatics.sdstate.edu/idep96/) and Subio (Subio Inc, Japan) platforms. A list of the enriched Kyoto Encyclopedia of Genes and Genomes pathways were obtained using the DAVID functional annotation web tool (https://david‐d.ncifcrf.gov).

We extracted total RNA using NucleoSpin RNA column kit (Takara Bio). After RNA isolation, we performed reverse transcription through the use of ReverTra ACE (TOYOBO, Osaka, Japan) following the manufacturer’s instructions. Afterward, we conducted quantitative real-time PCR with QuantStudio 7 system by using THUDERBIRD SYBR qPCR Mix kit (TOYOBO). We analyzed each sample in technical triplicates with the following protocol: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C. Each PCR analysis was completed by a melt curve cycle of 15 s at 95°C, 60 s at 60°C, and 15 s at 95°C. We analyzed all the data by using the standard curve method, and evaluated the relative amount of mRNAs compared to that of the house-keeping gene ACTB. We calculated the cycle threshold under the default settings by real-time sequence detection software (Applied Biosystems).
We used the following primer pairs for qPCR; EGR1 forward: 5′-AGCAGCACCTTCAACCCTCAGG-3′, reverse 5′-GAGTGGTTTGGCTGGGGTAACT-3′; PPARG forward: 5′-TGGTGACTTTATGGAGCCCAA-3′, reverse 5′-GGCAAACAGCTGTGAGGACTCAG-3′; DYRK1A forward: 5′-CCTTGCCATTGATATGTGGTCCC-3′, reverse 5′-GCAGGTGGAATACCCAGAACTTC-3′; ACTB forward: 5′-TCAAGATCATTGCTCCTCCTGAG-3′, reverse 5′-ACATCTGCTGGAAGGTGGACA-3′.

After a 48‐hour incubation period, cells from the direct and indirect coculture were harvested, and total RNA was extracted using a NucleoSpin RNA column kit (Takara Bio, Kusatsu, Japan). Reverse transcription and real‐time polymerase chain reaction analyses were done using a Light‐Cycler 480 with a Thunderbird SYBR quantitative polymerase chain reaction mix kit (Toyobo, Osaka, Japan) as previously described.¹⁹ The forward and reverse primer sets are presented in Table S1. Data were analyzed using the standard curve method. The cycle threshold was calculated using the default settings of the real‐time sequence detection software (ThermoFisher Scientific). Three independent experiments were conducted in each CF line.