Quantitative RT-PCR
TGGGCTTACTGACCAACCTG
To detect the mutation type in patient HSPCs, DNAs extracted using the QiampDNA mini kit (Qiagen, Cat. No. 51304) from patient granulocyte samples were standardized to 20 ng/ul and all samples tested at each time-point. Samples were excluded if they didn’t meet acceptance criteria of more than 60% mature granulocytes in DNA preparation, cell viability at freezing <95%, and less than 36 h in transit. A repeat blood sample was requested in these cases. JAK2V617F mutation analysis was performed as previously describedThe expression levels of nuclear estrogen receptors and membranal receptor in MPN cell lines were determined by qRT-PCR using standard methodologies. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat. No. 74104), and 500 ng was used in a reverse transcriptase reaction (High-Capacity cDNA Reverse Transcription kit, Applied Biosystems, Cat. No. 4368814) to generate cDNA for the template in quantitative real-time PCR reactions, which was performed using the PowerUp SYBR Green Master Mix (Applied Biosystems, Cat. No. A25742). Expression level was normalized to expression of endogenous control gene GAPDH. Primer sequence for human ESR1 is 5′ TGGGCTTACTGACCAACCTG 3′ (forward) and 5′ CCTGATCATGGAGGGTCAAA 3′ (reverse); for human GAPDH is 5′ GTCTCCTCTGACTTCAACAGCG 3′ (forward) and 5′ ACCACCCTGTTGCTGTAGCCAA 3′ (reverse).