Dnase treated rna

Total RNA was isolated from atrial and ventricular heart tissues using TRIzol reagent (15596-018; Invitrogen). cDNA was prepared from 1 μg of DNase treated RNA (4368813; Thermo Fisher Scientific). Primers targeting human DMPK exons 12–14 were used to assay transgene expression by real-time qPCR. Mouse Rpl4 was used as internal control for normalization. PCR reactions were carried out on an Applied Biosystems 7500 Fast Real-Time PCR System using PowerUp SYBR-Green PCR master mix (Thermo Fisher Scientific). Relative expression levels were determined by the 2^-ΔΔCt method. For analysis of alternative splicing events, primers annealing to flanking constitutive exons were designed. PCR products were resolved on a 5% polyacrylamide gel. Ethidium bromide stained RT-PCR bands were analyzed using Kodak Gel Logic 2000 and Carestream software. Percent spliced in (PSI) values were calculated using densitometry according to the equation: PSI = 100 X [Inclusion band/(Inclusion band + Skipping band)]. Primer sequences are provided in Supplemental Table 2 and Supplemental Table 3. See complete unedited blots in the supplemental material.

RNA was extracted from frozen leaf tissue following the protocol published by Oñate-Sánchez and Vicente-Carbajosa (2008) (link). 1–2 μg DNase treated RNA (ThermoFisher Scientific, MA, United States), were transcribed to cDNA using the Revert Aid RT (ThermoFisher Scientific, MA, United States) and oligo dT(18) primer following the manufacturer protocol. qPCR analysis was carried out using a Bio-Rad CFX Connect Real-Time PCR Detection System and SYBR green qPCR master mix (ChamQ Universal SYBR^® qPCR Master Mix, Absource, ger). Primer for gene expression analysis were published before (Richter et al., 2013 (link), 2016 (link)), and expression was normalized to SAND (AT2G28390) and calculated relative to the WT samples using the 2^–ΔΔC(t) method.