Quantitative reverse transcription polymerase chain reaction

To evaluate the delivery of pDNA, at 24 hours, cells cocultured with pGFPns-loaded CMPs were first washed with PBS, and pGFPns from cells was purified using the QIAprep Spin Miniprep Kit. A probe for the eGFP sequence was amplified through PCR, as detailed above, to confirm the delivery of pGFPns to HSPCs. At 72 hours, total RNA was isolated from the cells using the miRNeasy Micro Kit (Qiagen), and 0.5 μg of total RNA was reverse transcribed by the cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR assays for eGFP and GAPDH mRNA expression were performed with iTaq Universal SYBR Green Supermix (Bio-Rad). eGFP and GAPDH expression was quantified by the Livak method. To verify the level of gene knockdown of c-myb expression, total RNA was isolated at day 1 of coculture, and qRT-PCR analysis was performed with iTaq Universal SYBR Green Supermix. The gene expression level of c-myb was calculated with normalization to GAPDH. Primers used for qRT-PCR are listed in table S1

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