Figure 2.

Quantification of miRNAs in HEK293-derived AAV vector batches

MiRNAs were quantified by RT-qPCR in HEK293 cell extract (A) and in AAV vector batches produced in HEK293 mammalian cells (B–J). AAV8 full (B) and empty (C) particles were purified by CsCl, as well as AAV2 full (D) and empty (E) particles. Quantification was performed from duplicate lots of full AAV2 (F) and full AAV8 (G) purified by CsCl. AAV8 full (H) and empty (I) particles were purified from the same bulk by IA followed by CsCl gradient ultracentrifugation. Batch 9 corresponds to an AAV8 purified by IA (J). The same cassette (cytomegalovirus [CMV] promoter and enhanced green fluorescent protein [eGFP] transgene) was used for all AAV vectors. MicroRNAs were extracted in triplicate and quantified in duplicate by SYBR Green-based RT-qPCR. The relative quantity of each miRNA was determined using the ΔCt method. For AAV, data were normalized to account for culture and batch volumes.