Peqgreen dna rna dye

The quality of isolated DNA was analyzed by agarose gel electrophoresis [36 (link)]. Three microlitres of DNA was loaded onto a 2% agarose gel. Lambda DNA was used as a control as well as a marker for high-quality DNA. Nucleic acids were stained using peqGreen DNA/RNA dye (VWR, Germany). DNA quality was scored depending on molecular weight, grade of degradation, intensity of staining, and RNA contamination as detected by gel electrophoresis, with 1 high, 2 medium, and 3 low quality.

Tunicamycin (TM) is a drug typically used to induce ER stress which activates the UPR (Walter and Ron 2011 (link)). CHO K1 cells were treated with 0.5 μg/mL and 1 μg/mL TM (Sigma-Aldrich, no.T7765) for 2 h, 4 h, and 6 h. RNA of 10,000 cells was isolated using the RNeasy Mini Kit (Qiagen, no. 74104) according to the manufacturer’s instructions. Isolated RNA (200 ng) was used for reverse transcription (RT) with oligo dT and hexamer primers using QuantiTect Reverse Transcription Kit (Qiagen, no. 205311). For determination of XBP1 splicing (Lin et al. 2007 ), PCR amplification was performed with XBP1_CHO_fw (5′-TTGAGAGAGAAAACTCATGGC-3′) and rev (5′-GGGTCCAACTTGTCCAGAATGC-3′) primers in the conditions: 95 °C/5 min, 35× (95 °C/1 min, 58 °C/30 s, 72 °C/30 s) 72 °C/5 min. Separation of the PCR product was achieved on a 2.5% agarose gel using TAE buffer. Visualization of the band was enabled by peqGREEN DNA/RNA Dye (VWR, 732-3196) and 1 Kb Plus DNA Ladder (Thermo Fisher Scientific, no. 10787018) was used as marker. Intensity of the resulting bands was evaluated with the ImageJ software (Schneider et al. 2012 (link)).