Materials: The LIF Performance Test Mix (PN: 726022), Nano vials (PN 5043467) and pre-assembled EZ-CE Capillary Cartridge (PN A55625, Figure 1B) were from SCIEX, Framingham, MA. Urea (PN 29700), Nuclease-free water (PN AM9932), SYBR Green II RNA gel stain, 10,000x concentrate in DMSO (PN S7564), 10x DNase I buffer (PN AM8170G) were obtained from Thermo Fisher Scientific, Waltham, MA. Polyvinylpyrrolidone (PVP, PN 437190), benzonase (PN E1014-5KU), 0.5 M EDTA, pH 8.0 (PN E7889-100ML), Transcript RNA markers 0.2-10 kb (PN R7020) and 10x Tris Borate EDTA (TBE) buffer (PN 574795), Molecular Biology Grade, Amicon Ultra-0.5 centrifugal filter unit with MW cut off of 100 kDa (PN UFC510024) were from Millipore Sigma, St. Louis, MO. The 5 µm syringe filter (PN 4650) was from PALL Corporation, Port Washington, NY. Rainin LTS filter tips were from Mettler Toledo, Oakland, CA. QIAquick PCR purification Kit (PN 28104) and Proteinase K (PN 19131) were from Qiagen, Germantown, MD. Packaged AAV8 and AAV formulation buffer (1X PBS with 0.001% Pluronic F68) were from Vigene Biosciences, Rockville, MD. AAV5 and AAV2 were from Signagen, Rockville, MD.
Instrument and Software: A PA 800 Plus Pharmaceutical Analysis System (Figure 1A) equipped with LIF detector and solid-state laser with excitation wavelength at 488 nm and a 520 nm band pass emission filter were from SCIEX, Framingham, MA. Data acquisition and analysis were performed using 32 Karat™ Software 10.3.
LIF Calibration: To ensure consistent response of LIF detector throughout this study, the LIF detector was calibrated using LIF Calibration Wizard and Performance Test Mix (PN 726022) following the instructions in the PA 800 Plus System Maintenance Guide (PN A51964).
Sample Storage: AAV samples and the Sigma Transcript RNA Markers were aliquoted at 5 to 20 µL upon first thawing and stored at -80oC freezer to avoid multiple freeze-thaw cycles.
Sample Preparation for RNA Markers: The Sigma Transcript RNA markers were diluted in nuclease-free water to 1 ng/µL, heat-treated and loaded onto the instrument as described above for the AAV genome sample.
Preparation of Buffer Trays and Sample Trays: All solutions were pipetted with filter tips. Each “NF Water” vial was filled with 1.5 mL nuclease-free (NF) water. Waste vial was filled with 1 mL NF water. “Sep Buffer” and “TBE Buffer” vials were filled with 1.5 mL of separation buffer containing SYBR Green II dye or 1x TBE.