Quantifying Hematopoietic Progenitor Gene Expression

On GD 11.5, hematopoietic cells pooled from individual litters of vehicle- or TCDD-exposed fetuses were isolated and sorted directly into Trizol (Life Technologies) using c-Kit and DCF fluorescence to discriminate between hematopoietic progenitor cells with long- and short-term self-renewal potential, respectively. RNA was purified, quantified and processed as previously described (Ahrenhoerster et al. 2014 (link)). RNA was subjected to reverse transcription using a Tetro cDNA Synthesis kit (Bioline) with both anchored-oligo(dT) 18 priming and random hexamer priming options and was stored at –20°C until the day of the assay. Primers were selected using Universal ProbeLibrary v.2.5 for Mouse (Roche) and checked for specificity using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Gene names, accession numbers, and primer sequences are provided in Table S1. cDNA was used as a template in a 20-μL reaction consisting of 10 μmol of forward and reverse primers and 10 μL SensiFAST SYBR No-ROX (Bioline). Relative expression change was determined using the 2^–ΔΔC_T method with standardization to housekeeping genes. Samples were run in triplicate wells with at least two independent litters per treatment analyzed. Cycling conditions were 95°C for 2 min, followed by 45 cycles at 95°C for 5 sec, 60°C for 10 sec, and 72°C for 10 sec.