5-Aza Treatment Effects on Leukemia Cell Lines

One million human and murine leukemia cell lines were treated in vitro with 5 μM or 1 µM 5-Aza for 48 h in complete RPMI media, respectively. Total RNA from approximately 5 × 10⁵ cells was isolated using the Omega Biotek E. Z. N. A. Total RNA kit I (Norcross, GA, USA) with on column DNase I digestion of remaining genomic DNA. Total RNA was converted to cDNA using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit (using random primers) (Applied Biosystems; Waltham, MA, USA). cDNA was amplified and fluorescently labeled using the Bioland Scientific 2X qPCR Master Mix (Low Rox) (Paramount, CA, USA) containing SYBR Green in the QuantStudio3 Real Time PCR System (Thermofisher; Waltham, MA, USA). Experiments were run with “no template” controls to control for nucleic acid contamination in water or primers, no data was used if sample CTs fell within five CTs of the “no template” controls. Primer sequences were published previously [22 (link)].