Experion rna stdsense analysis kit

Total RNA was extracted from all in vitro inductions by combining Trizol^® (Invitrogen, Carlsbad, CA, USA) with the column-based E.Z.N.A. total RNA kit (Omega Biotek, Norcross, GA, USA) according to Wikström et al. [25 (link)]. RNA from PAXgene tubes was isolated combining the PAXgene Blood RNA Kit with Trizol extraction. In brief, the PAXgene Blood RNA protocol was followed with the exception that Trizol was added instead of BR2 binding buffer and the sample went through one round of Trizol/chloroform extraction before adding ethanol and transfer to the spin column.
RNA quantity and purity was estimated by spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies, Montchanin, DE, USA) and RNA quality index (RQI) was estimated using capillary gel electrophoresis (Experion RNA StdSense Analysis Kit, Bio-Rad Laboratories, Solna, Sweden). Total RNA (1.2 μg) from each sample were treated with RNAse-free DNAse I (Promega, Madison, WI, USA) followed by cDNA synthesis (GoScript Reverse transcription system; Promega, USA). Samples were diluted 5 × before storage at −20°C until qPCR analysis.

RNA extraction and cDNA synthesis were performed as previously described [18 (link)]. In brief, RNA was extracted by combining Trizol (Invitrogen, Carlsbad, CA, USA) with the column-based E.Z.N.A. total RNA kit (Omega Biotek, Norcross, GA, USA). RNA quantity and purity was estimated by spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies, Montchanin, DE, USA) and RNA quality index (RQI) was estimated to ≥ 9.8 using capillary gel electrophoresis (Experion RNA StdSense Analysis Kit, Bio-Rad Laboratories, Solna, Sweden). After treating 0.4–1 μg of RNA with RQ1 RNAse-free DNAse (Promega, Madison, WI, USA) cDNA was synthesized (GoScript Reverse transcription system; Promega) and diluted 5 × before storage at −20 °C.