Fast start essential dna green

Reverse transcription was performed on 1 μg of total RNA in a 20 μl reaction with the iScript cDNA synthesis kit (Bio-Rad Laboratories,170–8891). Quantitative real-time PCR was performed using a Roche Lightcycler 96 with Fast Start Essential DNA Green (Roche Diagnostics Corporation, 06-924-204-001) and primers from Integrated DNA Technologies, Inc. Primer efficiency was verified to be over 95% for all primer sets used. Quantification of mRNA was carried out via ΔΔCT analysis using GAPDH mRNA and the respective control condition for normalization. All real-time PCR primer sets were designed so the products would span at least one intron (>1kb when possible) to prevent detection of the pre-mRNA and/or DNA, and amplification of a single product was confirmed by agarose gel visualization and/or melting curve analysis (See S2 Table for sequences).

To assess the mRNA levels, S2-028 OrganoPlate cultures were exposed to gemcitabine (Sigma-Aldrich G6423) at day 3 for 72 h. Cells were lysed and RNA was purified using the TRIzol reagent (Thermo Fisher 15596026) with 7 µg Rnase-free glycogen (Thermo Fisher R0551) per sample added as a carrier, according to manufacturer’s protocol. Four to twenty chips were pooled into 1 sample, depending on the condition to compensate for the difference in cell density between the conditions. RNA concentration was measured using a NanoDrop (Thermo Fisher) and the samples were diluted to 30 ng/µL with RNase-free water. cDNA synthesis was performed using M-MLV reverse transcriptase (Thermo Fisher 28025013), according to manufacturer’s protocol. qPCR was performed using the FastStart Essential DNA Green (Roche 06402682001, Rotkreuz, Switzerland) using specific primers for the different MRP genes and using TBP as the housekeeping gene, see Table S1. The data were analyzed using the Roche LightCycler software version 1.1 and the 2^−ΔΔCt method, normalizing all values to the perfusion flow vehicle control per experiment.

Reverse transcription was performed on 10 μl m6A PolyA⁺ RNA from the MeRIP with the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). After diluting cDNA two-fold, quantitative real-time PCR was performed using a Roche Lightcycler 96 with Fast Start Essential DNA Green (Roche Diagnostics Corporation, Indianapolis, IN) and primers from Integrated DNA Technologies, Inc. (Coralville, Iowa). Primers used are listed in Supplementary Table 1. Primer efficiency was verified to be over 95% for all primer sets used. Quantification of mRNA from the MeRIP was carried out via ΔCT analysis against non-immunoprecipitated input RNA. All real-time PCR primer sets were designed so the products would span at least one intron (>1 kb when possible), and amplification of a single product was confirmed by agarose gel visualization and/or melting curve analysis.