Illustra rna extraction kit

For mRNA analysis, mRNA was isolated from cells using an Illustra RNA extraction kit (GE Healthcare, Ottawa, Canada) and reverse-transcribed using an iScript cDNA Synthesis Kit (Bio-Rad). iTaq Universal Probes Supermix (Bio-Rad) with PrimeTime Probes (IDT, Coralville, IA, USA) or SsoFast EvaGreen Supermix (Bio-Rad) with primers (Invitrogen) was used to run qPCR analysis on the 7500Fast system (Applied Biosystems, Foster city, CA, USA). For miRNA analysis, miRNA was isolated from cells using a miRNeasy miRNA isolation kit (Qiagen, Toronto, Canada) and TaqMan MicroRNA assays (Applied Biosystems) were performed according to manufacturer’s protocol to run qPCRs using the 7500Fast system. See Supp. Table 4 for miRNA assays and primers used for qPCR.

Total RNA from the OSE was extracted with the Illustra RNA extraction kit (GE Healthcare, Ottawa, Canada) according to the manufacturer’s protocol. RT was performed with 1000 ng RNA using an iScript cDNA Synthesis Kit (Bio-Rad, Mississauga, ON, Canada). Real-time PCR was performed with either Fast SYBR Green Master Mix (Invitrogen, Carlsbad, CA, US) for primers or iTaq universal probes supermix (Bio-Rad, Mississauga, ON, Canada) for probes. Each PCR reaction consisted of 5.5 μl of Fast SYBR Green Master Mix or iTaq, 2.5 μl of RNAse free water, 1 μl of cDNA sample, and 0.5 μl (10 pmol) of gene-specific primers/probes (Table S1). The qPCR was run using the 7500 Fast system assays (Applied Biosystems). To quantify relative gene expression, the cycle threshold (Ct) of target gene amplification was normalized to the expression level of a housekeeping gene (Ppia), according to the ratio, R = E^{Ct Ppia}/E^{Ct target}, where E is the amplification efficiency for each probe^{23 (link)}.