Iscript cdna synthesis system

Total RNA was extracted from cells using TRIzol (Invitrogen) and treated with DNase I (Ambion). One microgram of total RNA was used for first-strand DNA synthesis with the iScript cDNA Synthesis system (BioRad). qPCR was performed using human-specific primers and iTaq SYBR Green Universal Master Mix (Bio-Rad). The expression of each target mRNA relative to 18S rRNA was calculated based on the threshold cycle (Ct) as 2^−Δ(ΔCt), where ΔCt = Cttarget − Ct_18S and Δ(ΔCt) = ΔCt_test − ΔCt_control. Primer sequences are shown in Table S1.

Total RNA was isolated using TRIzol reagent (Invitrogen, CA) and subjected to reverse transcription with iScript cDNA Synthesis system (Bio-Rad, Hercules, CA). The abundance of TET mRNA was measured with real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad), as described previously.^{30 (link)} Primers used were 5′-acactagaacaagtagtggcaata-3′ (forward) and 5′-tttaagtttgggtcttggaggtct-3′ (reverse) for TET1, and 5′-ctgtaccacacggaggacact-3′ (forward) and 5′-gtagaggcgctggaataggac-3′ (reverse) for BK_Ca channel β1 subunit. Real-time PCR was performed in a final volume of 25 μL, and each PCR reaction mixture consisted of 500 nmol/L of primers and iQ SYBR Green Supermix containing 0.625 U hot-start Taq polymerase; 400 μmol/L each of dATP, dCTP, dGTP, and dTTP; 100 mmol/L KCl; 16.6 mmol/L ammonium sulfate; 40 mmol/L Tris-HCl; 6 mmol/L MgSO₄; SYBR Green I; and 20 nmol/L fluorescing and stabilizers. The following protocol was used for real-time PCR: 95 °C for 5 minutes, followed by 45 cycles of 95 °C for 30 seconds and annealing for 30 seconds at 52 °C and for 45 seconds at 72 °C. GAPDH was used as an internal reference, and serial dilutions of the positive control were performed on each plate to create a standard curve for the quantification. PCR was performed in triplicate, and threshold cycle numbers were averaged for each sample.